Platelets from WAS patients show an increased susceptibility to ex vivo phagocytosis

The thrombocytopenia of Wiskott-Aldrich syndrome (WAS) is thought to be due to both reduced platelet production and accelerated platelet consumption. We have previously demonstrated that platelets from WASP-deficient mice are consumed more rapidly in vivo than are WT platelets, and that opsonization accelerates their uptake by bone marrow- derived macrophages more than it does that of WT platelets. Here we asked whether platelets from WAS patients show similar features. We show that ex vivo phagocytosis by activated THP-1 cells of DIO-labeled platelets from a series of WAS or XLT patients is increased in comparison to that of normal control platelets. Using a numerical analysis method, we distinguish this effect from a concurrent effect on the amount of detectable fluorescent signal transferred to the macrophage per phagocytosed platelet. We show that the latter quantity is reduced by platelet WASP deficiency, as might be expected if the fluorescence transferred from these smaller platelets is more rapidly quenched. We are unable to detect a differential effect of opsonization with anti-CD61 antibody on the uptake of WASP(-) vs. WT platelets. However, the high probability of phagocytosis per adsorbed WASP(-) platelet could limit the sensitivity of the assay in this case. We also see no effect of sera from WAS patients on the uptake of normal control platelets, suggesting that in vivo opsonization is not the cause of increased uptake of WASP(-) platelets. Finally, we show little, if any, increase in the reticulated platelet fraction in WAS patients, suggesting that impaired production of reticulated platelets contributes to the thrombocytopenia. Our findings suggest that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. They also demonstrate the feasibility of routinely performing functional assays of phagocytosis of small numbers of platelets obtained at remote locations, a method which should be applicable to the study of other types of thrombocytopenia such as ITP.