Objective-Our objective was to determine a mechanism for the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS).Materials and Methods-Consumption rates of WAS protein (WASP)( −) and wild-type (WT) platelets were measured by injection of 5-chloromethylfluorescein diacetate (CMFDA)-labeled platelets into WT or WASP(−) recipients, and by in vivo biotinylation. Platelet and reticulated platelet counts were performed using quantitative flow cytometry. Bone marrow megakaryocyte number and ploidy was assessed by flow cytometry. Phagocytosis of CMFDA-labeled, opsonized platelets was assessed using bone marrow-derived macrophages. Serum antiplatelet antibodies were assayed via their binding to WT platelets.Results-CMFDA-labeled WASP(−) platelets are consumed more rapidly than WT platelets in either WT or WASP(−) recipients. In vivo biotinylation studies corroborate these findings and show a normal consumption rate for WASP(−) reticulated platelets. The number of reticulated platelets is reduced in WASP(−) mice, but a significant number of the mice show an increased proportion of reticulated platelets and more severe thrombocytopenia. Sera from some of the latter group contain antiplatelet antibodies. Compared to WT platelets, WASP(−) platelets opsonized with anti-CD61 or 6A6 antibody are taken up more rapidly by bone marrow-derived macrophages. In vivo consumption rates of WASP(−) platelets are more accelerated by opsonization than are those of WT platelets.Conclusion-Both rapid clearance and impaired production contribute to the thrombocytopenia of murine WAS. Increased susceptibility of opsonized WASP(−) platelets to phagocytosis leads to increased in vivo clearance. This correlates with a higher incidence of individuals with an elevated fraction of reticulated platelets, a more severe thrombocytopenia, and antiplatelet antibodies. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe Wiskott-Aldrich syndrome (WAS) is an X-linked recessive condition of variable penetrance, classically described as a triad of immunodeficiency, eczema, and thrombocytopenia [1,2]. It is caused by mutations that reduce the level of the WAS protein (WASP). WASP is a 54-kDa polypeptide that is expressed primarily, but not exclusively [3,4], in hematopoietic cells. WASP transmits and integrates signals arising at the cell membrane that result in actin polymerization. This can, in turn, have multiple effects, including but not limited to changes in cell shape and motility. While its function in T cells and macrophages has been studied in some detail [5,6], its biological role in platelets is not clear.WAS patients can have severe thrombocytopenia, with platelet counts ranging from 10,000 to 50,000 per uL in one series [7]. In some cases, the thrombocytopenia is the predominant clinical abnormality. These cases, formerly termed X-linked thrombocytopenia, are frequently due to missense mutations in the first four exons of the gene [8], and can show a fluctuating course resembling immune t...

The Wiskott-Aldrich Syndrome is a congenital X-linked immunodeficiency caused by mutations in the WASP gene. The most common clinical presentation is recurrent infection, eczema, and thrombocytopenia. Autoimmune disease occurs in as much as 70% of WAS patients. WASP functions to transmit and integrate signals that originate at the cell membrane and result in actin polymerization. The latter in turn augments signal transduction downstream of cell surface ligands such as the T-cell receptor and macrophage integrins. Dysfunction of these signal transduction pathways is thought to result in the immunodeficiency. The mechanism by which WASP deficiency results in thrombocytopenia, however, is not well understood. Although WASP(−) platelets are smaller than normal, no cytoskeletal defect has been consistently observed in them. While platelet aggregation abnormalities have been reported, it is not known whether they contribute significantly to the hemorrhagic complications seen in severely thrombocytopenic WAS patients. WASP(−) platelets are consumed more rapidly in vivo than are normal platelets, both in normal volunteers and in WAS patients. Platelet production rate may be reduced as well. Splenectomy improves platelet counts in WAS patients, but the subsequent incidence of ITP is high (23% in one study). WAS patients with low but detectable levels of WASP can show a fluctuating thrombocytopenia similar to the course of ITP. These findings suggest that autoimmunity could contribute to the thrombocytopenia of WAS. A murine model of WAS shows a milder thrombocytopenia (approximately 50% of normal) and normal platelet size. We have shown that WASP(−) murine platelets are consumed more rapidly than WT platelets in either WT or WASP(−) recipients. Their in vivo consumption rate is more affected by antibody opsonization than is that of WT platelets, a finding we have corroborated with ex vivo phagocytosis studies. A subset of WASP(−) mice show a more severe thrombocytopenia with an increased fraction of reticulated platelets (RP), suggesting the presence of antiplatelet antibodies. To test this, we have optimized a flow cytometric assay for serum antiplatelet antibodies by using target platelets that are briefly formalin fixed, and using target platelets from muMT(−/−) mice, which lack background levels of surface antibodies. Using this assay, we have detected serum anti-platelet antibodies in a subset of WASP(−) and WASP(−)CD47(−/−) mice. We have not detected antibodies in WT(B6) mice or in CD47(−/−) mice. We are unable to detect these antibodies with non-fixed target platelets. Use of fixed WT target platelets significantly reduces the sensitivity of the assay. The antibodies are predominantly of the IgG class. Their in vivo significance is supported by the finding that they are more frequent in WASP(−) mice with increased RP (4 of 5 tested) than in those with normal RP (1 of 7 tested). Also, antibody expressing (Ab+) mice show more rapid consumption of CMFDA-labeled WASP(−) platelets than do Ab-mice. To determine whether these antibodies have the differential effect on in vivo WASP(−) platelet consumption that we previously observed after ex vivo opsonization, we simultaneously quantified the consumption rate of WT and WASP(−) platelets in antibodyexpressing (Ab+) and antibody-negative (Ab−) WASP(−) mice. We did this by labeling the two platelet preparations with different fluorescent markers (CMFDA and BMQC). We verified that the fluorescent markers had no differential effect on platelet consumption in vivo. We then found more rapid consumption of both WT and WASP(−) platelets in Ab(+) mice. In some of the latter, WASP(−) platelet consumption is enhanced relative to that of WT platelets. These results suggest that WASP deficient mice, and WAS patients, may be both more prone to develop antiplatelet antibodies and, in some cases, more susceptible to their effects on platelet consumption.