A possible defect in mitochondrial ATP resynthesis in platelets has been used for detection of Wiskott-Aldrich syndrome carriers (Shapiro et al, 1978). The detection was based on an abnormal adrenaline-induced platelet aggregation under conditions that only the mitochondria provide metabolic energy. We evaluated the test and found false negative and false positive results which raised doubts about the applicability of the test and the nature of the underlying defect. Direct analysis of mitochondrial ATP regeneration in patients and carriers showed an impaired mitochondrial energy production which was insufficient to maintain ATP homeostasis when glycolytic energy production was inhibited. Also abnormal was the fall in metabolic ATP concentration during stimulation with adrenaline and especially with thrombin when the platelets were incubated in glucose-free medium. These data provide direct evidence for a regulation defect in mitochondrial ATP resynthesis in platelets of patients and carriers with Wiskott-Aldrich syndrome.
WAS is a severe, X-linked disorder, characterized by eczema, immunodeficiency and an increased bleeding tendency caused by thrombocytopenia and platelet malfunction. There is a diminished epinephrine-induced aggregation response and an abnormal mitochondrial CO2 production during platelet activation. From this, Shapiro et al (The Lancet 1978) concluded that WAS-platelets have a defect in mitochondrial ATP regeneration, which could be employed for detection of WAS- carriers, who are clinically normal and have only minor platelet defects. The test consists of an epinephrine-induced aggregation in the presence of an inhibitor of glycolytic ATP production (deoxyglucose, 2 DG), and showed impaired second wave aggregation in obligate carriers but not in normal controls. We tested 4 unrelated obligate WAS-carriers and found impaired aggregations in all. Five out of 7 female relatives also showed aggregation abnormalities, suggestive for WAS-carriership. However, in 8 out of 15 normal controls (males and females) the test was also positive. The nature of a possible defect in mitochondrial ATP supply was further studied in gel-filtered platelets by analyzing the metabolic ATP level before and during epinephrine-induced aggregation in the presence of inhibitors of glycolysis and glycogenoly- sis and during incubation in substrate-depleted medium. These studies showed that mitochondrial energy generation depended on sugar supply either from glycolysis or glycoge- nolysis and was unable to maintain a normal metabolic ATP level when these pathways were inhibited. Incubation with 2DG led to a fall in metabolic ATP and - consequently - to an impaired epinephrine-induced aggregation. The fall of metabolic ATP (2DG present) was much steeper in platelets from 2 unrelated WAS-patients than in cells from normal controls; most (but not all) obligate carriers showed intermediate values. It is concluded that the impaired epinephrine-induced aggregation in the presence of 2DG in WAS reflects disturbances in ATP homeostasis, which are consistent with a mitochondrial defect.
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