Thrombocytopenia Of Wiskott-Aldrich Syndrome Research Articles

Assessment of Immature Platelet Fraction in the Diagnosis of Wiskott-Aldrich Syndrome.

Children with Wiskott–Aldrich syndrome (WAS) are often first diagnosed with immune thrombocytopenia (ITP), potentially leading to both inappropriate treatment and the delay of life-saving definitive therapy. WAS is traditionally differentiated from ITP based on the small size of WAS platelets. In practice, microthrombocytopenia is often not present or not appreciated in children with WAS. To develop an alternative method of differentiating WAS from ITP, we retrospectively reviewed all complete blood counts and measurements of immature platelet fraction (IPF) in 18 subjects with WAS and 38 subjects with a diagnosis of ITP treated at our hospital. Examination of peripheral blood smears revealed a wide range of platelet sizes in subjects with WAS. Mean platelet volume (MPV) was not reported in 26% of subjects, and subjects in whom MPV was not reported had lower platelet counts than did subjects in whom MPV was reported. Subjects with WAS had a lower IPF than would be expected for their level of thrombocytopenia, and the IPF in subjects with WAS was significantly lower than in subjects with a diagnosis of ITP. Using logistic regression, we developed and validated a rule based on platelet count and IPF that was more sensitive for the diagnosis of WAS than was the MPV, and was applicable regardless of the level of platelets or the availability of the MPV. Our observations demonstrate that MPV is often not available in severely thrombocytopenic subjects, which may hinder the diagnosis of WAS. In addition, subjects with WAS have a low IPF, which is consistent with the notion that a platelet production defect contributes to the thrombocytopenia of WAS. Knowledge of this detail of WAS pathophysiology allows to differentiate WAS from ITP with increased sensitivity, thereby allowing a physician to spare children with WAS from inappropriate treatment, and make definitive therapy available in a timely manner.

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.

Platelets From WAS Patients Are More Susceptible Than Controls to Phagocytosis by Activated THP-1 Cells

Abstract 2222The thrombocytopenia associated with the Wiskott-Aldrich Syndrome (WAS) is thought to be due to the combined effects of impaired platelet production and accelerated platelet consumption. In a murine model of WAS, we have previously demonstrated that platelet consumption is accelerated. Also, we have shown that antibody opsonization accelerates both in vivo consumption and ex vivo phagocytosis of murine WASP(-) platelets in comparison to opsonized WT platelets. Based on these findings, we tested the susceptibility of platelets from WAS patients to ex vivo phagocytosis by activated THP-1 cells. We used a lipophilic fluorescent marker (DIO) to label platelets, and distinguished between platelet uptake and adsorption with a fluorescent anti-CD61 antibody. In comparison to previous methods using CMFDA-labeled platelets, use of DIO resulted in a tenfold reduction in the number of platelets needed per assay. This in turn allowed us to perform ex vivo phagocytosis studies with the very small number of platelets available for study in peripheral blood specimens from thrombocytopenic WAS patients. We report that untreated platelets from WAS patients are taken up more rapidly than control platelets by activated THP-1 cells. Specifically, the fraction of macrophages demonstrating DIO uptake, and showing no adsorbed platelets is consistently increased in comparison to cells showing adsorbed platelets (figure 1). Using a numerical analysis method, we distinguish the effect of WASP deficiency on the probability of phagocytosis per adsorbed platelet (p) from effects on the fluorescence intensity imparted to the macrophage per internalized platelet (alpha) and from variation in the platelet/macrophage ratio (m). This was done by predicting the type of results shown in figure 1 for approximately 32,000 possible combinations of p, alpha, and m, and finding among the possible combinations those for which the predicted results best fit our observations. We validated the sensitivity of the method to changes in p (via the use of opsonized vs. non-opsonized platelets), m (via changing the platelet/macrophage ratio) and alpha (via changing the mean fluorescence intensity of the platelets). Applied to the data in figure 1, the numerical analysis method demonstrates an increased p value for each of the WAS patients studied, and a reduced alpha value (as might be expected for smaller platelets) in 5 of 6 studies (figure 2). Opsonization with murine anti-CD61 antibody did not accelerate uptake of WAS platelets in comparison to controls. However, we observed significantly increased levels of surface IgM, and possibly IgG, on platelets from WAS patients. In addition to inhibiting opsonization ex vivo, this level of surface antibody could contribute to accelerated phagocytosis of platelets in clinical WAS in the same way that ex vivo opsonization augments the in vivo clearance and ex vivo phagocytosis of murine WASP(-) platelets. Our results provide additional support for the role of accelerated platelet consumption in generating the thrombocytopenia of WAS. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The thrombocytopenia of WAS: a familial form of ITP?

In the first report of the concurrent immunodeficiency, thrombocytopenia, and eczema that we now call the Wiskott-Aldrich Syndrome (WAS), Alfred Wiskott asked whether it could be a familial form of Werlhof's disease (now called ITP). This review summarizes what is known about platelet production, consumption, and function in clinical and murine WAS. Both platelet production and consumption are affected by WASP deficiency. Likely molecular mechanisms have been identified for the former process, but remain problematic for the latter. Recent data in a murine model suggest that WASP deficiency could increase both the incidence of antiplatelet antibodies and susceptibility to their enhancement of platelet consumption. Wiskott's original speculation about the relationship between WAS and ITP may need to be reconsidered.

Increased Extra-Splenic Platelet Turnover Contributes to the Thrombocytopenia of Murine WAS.

The Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive condition manifesting as variable degrees of immunodeficiency and thrombocytopenia. The strong clinical improvement in platelet count seen in WAS patients after splenectomy suggests that increased platelet consumption is a major contributor to the thrombocytopenia. Here we show that splenectomy improves but does not normalize platelet count in a murine (C57Bl/6) model of WAS, suggesting (a) that murine WAS parallels clinical WAS in this regard, and (b) that either increased extrasplenic consumption or diminished platelet production (or both) contributes to the thrombocytopenia. We quantified platelet turnover by labeling WAS and WT platelets with CMFDA and following their disappearance after injection into WT recipients. Platelet loss rates in this model fit best to a simple exponential decay curve. We found that WAS platelets turn over significantly faster than WT (survival times: 56 hr (WT) vs 40 hr (WAS), p <.01). By comparison, we have observed a 42% reduction in platelet count in WAS mice (508K/ul, vs 881K/ul for age matched WT littermates, p<.01). WAS mice also show increased splenic extramedullary hematopoiesis, and increased megakaryocyte counts in bone marrow as assayed by flow cytometry. These results suggest that both increased platelet consumption and reduced thrombopoiesis contribute to the thrombocytopenia of WAS in this system, while megakaryopoiesis may not be inhibited. To determine the site of increased consumption of WAS platelets, we repeated the turnover studies in splenectomized C57Bl/6 mice. The results fit a two-hit gamma function, with estimated lifespans of 65 hr (WT) vs 53 hr (WAS)(p <. 01), indicating that splenectomy improved but did not normalize turnover of WAS platelets. The data suggests that the spleen is not the site of increased platelet consumption in WAS mice. To further delineate the role played by splenic platelet consumption in the thrombocytopenia of WAS, we crossed C57Bl/6 WAS mice for three generations onto the reportedly hypersplenic mouse strain C3H. Suprisingly, WAS (C3H) males showed no significant thrombocytopenia. Nonetheless, we found that WAS (C3H) platelets turn over more rapidly than WT platelets in C3H mice (estimated lifespans 61 hr (WT) vs 42 hr (WAS), p <.01), despite the fact that in comparison to C57Bl/6J (but not C57Bl/6Nhsd) mice, C3H mice do not in fact have significantly larger spleens. These results suggest that WAS (C3H) mice are able to compensate for increased platelet turnover rates by increasing platelet production appropriately, and imply that WAS (C57Bl/6) mice are not. Taken together, these studies suggest that the thrombocytopenia of murine WAS (C57Bl/6) is due to both increased extrasplenic platelet consumption and deficient platelet production. The inter-strain variation in WAS phenotype we report here reproduces the marked inter- and intra-kindred phenotypic variation seen in clinical WAS. [Display omitted]

The Thrombocytopenia of Wiskott Aldrich Syndrome Is Not Related to a Defect in Proplatelet Formation

The Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease characterized by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections. The gene responsible for WAS was recently cloned. Although the precise function of WAS protein (WASP) is unknown, it appears to play a critical role in the regulation of cytoskeletal organization. The platelet defect, resulting in thombocytopenia and small platelet size, is a consistent finding in patients with mutations in the WASP gene. However, its exact mechanism is unknown. Regarding WASP function in cytoskeletal organization, we investigated whether these platelet abnormalities could be due to a defect in proplatelet formation or in megakaryocyte (MK) migration. CD34+ cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked thrombocytopenia (XLT) and cultured in serum-free liquid medium containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor (SCF) to study in vitro megakaryocytopoiesis. In all cases, under an inverted microscope, normal MK differentiation and proplatelet formation were observed. At the ultrastructural level, there was also no abnormality in MK maturation, and normal filamentous MK were present. Moreover, the in vitro produced platelets had a normal size, while peripheral blood platelets of the same patients exhibited an abnormally small size. However, despite this normal platelet production, we observed that F-actin distribution was abnormal in MKs from WAS patients. Indeed, F-actin was regularly and linearly distributed under the cytoplasmic membrane in normal MKs, but it was found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from WAS patients normally migrated in response to stroma-derived factor-1 (SDF-1), and actin normally polymerized after SDF-1 or thrombin stimulation. These results suggest that the platelet defect in WAS patients is not due to abnormal platelet production, but instead to cytoskeletal changes occuring in platelets during circulation.

The Thrombocytopenia of Wiskott Aldrich Syndrome Is Not Related to a Defect in Proplatelet Formation

AbstractThe Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease characterized by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections. The gene responsible for WAS was recently cloned. Although the precise function of WAS protein (WASP) is unknown, it appears to play a critical role in the regulation of cytoskeletal organization. The platelet defect, resulting in thombocytopenia and small platelet size, is a consistent finding in patients with mutations in the WASP gene. However, its exact mechanism is unknown. Regarding WASP function in cytoskeletal organization, we investigated whether these platelet abnormalities could be due to a defect in proplatelet formation or in megakaryocyte (MK) migration. CD34+ cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked thrombocytopenia (XLT) and cultured in serum-free liquid medium containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor (SCF) to study in vitro megakaryocytopoiesis. In all cases, under an inverted microscope, normal MK differentiation and proplatelet formation were observed. At the ultrastructural level, there was also no abnormality in MK maturation, and normal filamentous MK were present. Moreover, the in vitro produced platelets had a normal size, while peripheral blood platelets of the same patients exhibited an abnormally small size. However, despite this normal platelet production, we observed that F-actin distribution was abnormal in MKs from WAS patients. Indeed, F-actin was regularly and linearly distributed under the cytoplasmic membrane in normal MKs, but it was found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from WAS patients normally migrated in response to stroma-derived factor-1α (SDF-1α), and actin normally polymerized after SDF-1α or thrombin stimulation. These results suggest that the platelet defect in WAS patients is not due to abnormal platelet production, but instead to cytoskeletal changes occuring in platelets during circulation.

Wiskott-Aldrich syndrome: current research concepts.

Wiskott-Aldrich syndrome: current research concepts.