Rapid platelet turnover in WASP(−) mice correlates with increased ex vivo phagocytosis of opsonized WASP(−) platelets

Abstract

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...

Calcium- and integrin-binding protein 1 regulates megakaryocyte ploidy, adhesion, and migration

“…Therefore, we assessed platelet clearance in both WT and Cib1 Ϫ/Ϫ mice using an in vivo biotinylation technique. 21 As with platelet production, the percentage of platelets cleared was unaltered on Cib1 deletion ( Figure 1B; supplemental Figure 2). Increased platelet count, combined with normal platelet production and clearance, suggests that, in terms of platelet regulation, Cib1 Ϫ/Ϫ mice simply operate at a higher baseline than the WT.…”

Pathogenesis and management of inherited thrombocytopenias: rationale for the use of thrombopoietin-receptor agonists

“…WASp is expressed exclusively in hematopoietic cells and has a key role in actin polymerization and reorganization of actin cytoskeleton. Studies on WASp-null mice and WAS patients demonstrated that defective platelets are cleared at a faster rate by macrophages [70][71][72]. Anti-platelet autoantibodies could contribute to this process [73].…”

The contribution of mouse models to the understanding of constitutional thrombocytopenia

“…Total inactivation reproduces the thrombocytopenia, which is moderate to severe depending on the genetic background and the individual, although microcytosis is absent. [35][36][37] Mouse models of constitutional thrombocytopenia haematologica | 2016; 101 (8) 901 Absence of WASp resulted in ectopic platelet release. 37 By relaying the signalling induced by collagen I and favoring the migration of megakaryocytes to sinusoids, WASp plays a key role in preventing the premature release of platelets in the bone marrow compartment.…”

“…37 In addition, this model proved to be useful in showing that the increased platelet consumption resulted from both intrinsic platelet factors and extrinsic factors, due to an increased susceptibility of these mice to develop platelet antibodies with subsequent opsonization and phagocytosis of platelets. 35,36 The development of "knock-in" mouse models has allowed one to explore the mechanisms governing the activation and stability of WASp, including the important role of phosphorylation at the Y293 single site (the murine equivalent of the Y291 site in human WASp). This phosphorylation site is critically involved in the activation of WASp in numerous cellular responses including proliferation, phagocytosis and especially the assembly of adhesion and chemotaxis structures.…”