Tyrosine kinase signaling regulates Wiskott-Aldrich syndrome protein function, which is essential for megakaryocyte differentiation.

Platelets are produced from megakaryocytes differentiated from megakaryoblasts, but the differentiation mechanism still remains unknown. Here, we demonstrate that a tyrosine kinase signaling regulates Wiskott-Aldrich syndrome protein (WASP), which is essential for megakaryocyte differentiation. MEG-01 megakaryoblastic cells differentiate into large multinucleated megakaryocyte-like cells characterized by microvesicle formation with a protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate. With parallel to the differentiation, WASP begins to accumulate at microvesicles with actin filaments and associates with tyrosine-phosphorylated Shc, the phosphorylation of which is induced. Moreover, a tyrosine kinase inhibitor, herbimycin A, blocked not only phosphorylation of Shc but also microvesicle formation without affecting cell enlargement and multinucleation, whereas a PKC inhibitor, H-7, completely inhibited all these processes. Because WASP also binds to Ash/Grb2 SH3 domains and the association of Ash/Grb2 and Shc is induced by 12-O-tetradecanoylphorbol 13-acetate treatment, a signaling pathway, PKC-tyrosine kinase-Shc-Ash/Grb2-WASP, is suggested for regulating megakaryocyte differentiation. In addition, WASP antisense oligonucleotide treatment abolishes both microvesicle formation and gathering of actin filaments. These data clearly show that WASP controls the assembly of actin filaments required for microvesicle formation downstream of PKC-tyrosine kinase.

Evidence implicating calpain (Ca(2+)-dependent neutral protease) in the destructive thrombocytopenia of the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is an inherited platelet/T-lymphocyte disease characterized by small platelets, thrombocytopenia and immunodeficiency. Because degradative events have a significant role, we directly examined calpain (Ca(2+)-dependent neutral protease), a prominent protease in the affected cells, by functional and antigenic quantitation. Calpain activity in platelets of seven WAS patients was decreased to 59 +/- 3.7% (P < 0.01) relative to platelets of 11 normals. Platelets of two patients with immune thrombocytopenia had normal calpain activity. By immunoblotting, mu-procalpain, the mu-calpain species in resting (unstimulated) blood cells, was decreased in platelets of nine WAS patients to 58 +/- 14.6% (P < 0.01) relative to paired normals. In contrast, mu-procalpain levels in lymphocytes of seven WAS patients did not differ from normal lymphocytes. Normal platelets and lymphocytes have different mechanisms for Ca(2+)-dependent mu-procalpain activation. On addition of ionophore and Ca2+ to stirred platelets, 80kD mu-procalpain was rapidly (0.5 min) and quantitatively converted to 76 kD active mu-calpain; this process was the same in WAS platelets. In lymphocytes, mu-procalpain activation was slow, only partially complete (40 min), and the active species was 78 kD. The marked depletion of calpain in WAS platelets demonstrated in this study may result from inappropriate stimulation of platelets and be related to the severe thrombocytopenia that characterizes this disease.

X-chromosome inactivation in the Wiskott-Aldrich syndrome: a marker for detection of the carrier state and identification of cell lineages expressing the gene defect.

Recently developed techniques for the direct analysis of DNA have made possible the determination of patterns of cellular X-chromosome inactivation. These techniques provide a potential method for carrier detection for several X-linked human disorders in which obligate carriers show nonrandom X inactivation. By using restriction fragment length polymorphic (RFLP) gene-specific probes in conjunction with methylation-sensitive enzymes, we have characterized the patterns of X-chromosome inactivation in cell subsets from females belonging to 10 kindreds segregating for the X-linked immune deficiency disorder Wiskott-Aldrich syndrome (WAS). We show that selective inactivation of the X chromosome distinguishes obligate WAS carriers from noncarrier females and constitutes a valuable marker of the WAS carrier state. Selective inactivation phenomena were observed in the monocytes and T and B lymphocytes of obligate carriers, implying that the WAS gene defect is expressed in each of these cellular lineages. In conjunction with the use of linked DNA markers, RFLP-methylation analysis should render carrier detection feasible for the majority of females from WAS families. The results of such analyses also provide an initial step toward identifying the cellular level and molecular basis for WAS.

Allelic exclusion of glucose-6-phosphate dehydrogenase in platelets and T lymphocytes from a Wiskott-Aldrich syndrome carrier.

An obligate carrier of the Wiskott-Aldrich syndrome (WAS) who was also heterozygous for the A and B types of X-linked glucose-6-phosphate dehydrogenase was found. With her it became possible to determine whether allelic exclusion occurs in particular cell-types of the WAS carrier. If so, the remaining cells of a particular cell-type would express only the normal X chromosome and only one glucose-6-phosphate dehydrogenase isoenzyme would be demonstrable. This carrier had only the B isoenzyme of glucose-6-phosphate dehydrogenase in platelets and thymus-derived T lymphocytes, although both isoenzymes A and B were present in erythrocytes and neutrophils. These findings suggest that selection against the WAS gene occurs in platelets and thymus-derived T lymphocytes and that the defects associated with WAS expressed in these cell-types may be implicated in the genesis of the Wiskott-Aldrich syndrome.