Rapid In Vivo Consumption and Ex Vivo Phagocytosis of WASP(−) Platelets.

Patients with the Wiskott-Aldrich Syndrome (WAS) have thrombocytopenia and increased platelet consumption rates, and may have reduced platelet production rates. WASP(−) mice have been reported to have only a mild thrombocytopenia. We find that when fully crossed onto the C57Bl/6J background, WASP(−) mice have a >50% reduction in platelet counts. Ex vivo labeled WASP(−)platelets are consumed 2 to 4-fold faster than are WT platelets in WT mice, and with exponential rather than linear kinetics. Clearance rates of WASP(−) platelets in WT mice, and vice versa, indicate that rapid consumption is due to factors both intrinsic and extrinsic to platelets. In vivo biotinylation demonstrates consumption rates comparable to those seen with ex vivo labeling, and shows a normal rate of consumption of WASP(−) reticulated (immature) platelets. Reticulated platelet counts are reduced, indicating that their production rate is reduced. Megakaryocytes are increased in spleen and bone marrow, and in the latter their ploidy distribution is normal, suggesting that impaired platelet production occurs at the level of thrombopoiesis. The absolute turnover rates of mature and reticulated platelets, however, indicate that maturation of the latter can account for only a fraction of the former’s turnover in either WT or WASP(−) mice. A subset of WASP(−) mice show an increased fraction of reticulated platelets and more severe thrombocytopenia, and some members of this subset also express serum anti-platelet antibodies. CMFDA-labeled WASP(−) platelets opsonized with anti-CD61(IgG1) antibody are more susceptible to ex vivo phagocytosis by bone marrow derived macrophages (BMDM) than WT platelets, and as susceptible as CD47(−/−) platelets. After opsonization with 6A6(IgG2b) antibody, WASP(−) platelets are also taken up more rapidly than WT platelets by BMDM, but less rapidly than are CD47(−/−) platelets. The in vivo consumption rate of WASP(−) platelets in WT recipients is more accelerated by opsonization with anti-CD61 antibody than is that of WT platelets. Increased phagocytosis is not due to (A) altered levels of the targeted antigens or of CD47 (B) increased exposure of phosphatidyl serine, or (C) antibody-induced activation as assayed by CD62P expression. Increased phagocytosis is not due to selective sensitivity to IgG1 vs IgG2b antibodies, as opsonization with an engineered 6A6(IgG1) antibody leads to reduced phagocytosis for both CD47(−/−) and WASP(−) platelets. Anti-CD61 opsonized platelets deficient in both WASP and CD47 show markedly increased ex vivo phagocytosis compared to platelets deficient in either protein, suggesting that platelet WASP does not function to amplify signals from platelet CD47 through macrophage SIRP-alpha. These results raise the possibility that the binding of low affinity or low titer antibodies that might have no effect on WT platelets could cause thrombocytopenia when the platelets lack WASP. Alternatively, rapid phagocytosis of opsonized WASP(−) platelets could promote a self-reinforcing cycle of increased host antigen presentation and increased immune responses to host antigens.