WASP and N-WASP in human platelets differ in sensitivity to protease calpain

Abstract

Mutations of Wiskott-Aldrich syndrome protein (WASP) underlie the severe thrombocytopenia and immunodeficiency of the Wiskott-Aldrich syndrome. WASP, a specific blood cell protein, and its close homologue, the broadly distributed N-WASP, function in dynamic actin polymerization processes. Here it is demonstrated that N-WASP is expressed along with WASP, albeit at low levels, in human blood cells. The presence of approximately 160 nmol/L rapidly acting N-WASP molecules may explain the normal capacity of WASP-negative patient platelets for early agonist-induced aggregation and filopodia formation. Ex vivo experiments revealed a significant difference between WASP and N-WASP in sensitivity to calpain, the Ca ؉؉ -dependent protease activated in agonist-stimulated platelets. IntroductionWiskott-Aldrich syndrome protein (WASP) and its close homologue, the broadly distributed N-WASP, members of the WASP/ SCAR protein family (reviewed in 1 ), are cytoplasmic proteins thought to mediate dynamic cytoskeletal rearrangements by nucleating actin filament formation in response to specific stimuli. 2 N-WASP, which was discovered in brain, has broad tissue distribution. 3 WASP, in contrast, is exclusive to blood cells. 4,5 Mutations of WASP lead to the Wiskott-Aldrich syndrome (WAS), a severe blood cell disease that includes immunodeficiency of variable severity and profound thrombocytopenia (reviewed in 6 ). A recent genotype-phenotype study established that WAS patients with severe immune disease (nonsense and frameshift mutations) have WASP-negative leukocytes and that patients with mild immune disease (missense mutations) have decreased WASP levels in leukocytes. 7 In contrast, 18 patients with diverse mutations all had WASP-negative platelets, providing an apparent explanation for the uniform severity of platelet dysfunction in this disease.Although substantial inroads have been made in delineating the biochemistry, regulation, and signaling capability of N-WASP and WASP, the pathologic steps leading to the dysfunction of WAS blood cells remain undefined. Neither is it known how blood cells of patients retain a substantial number of normal cytoarchitectural responses despite the absence or deficit of WASP. For example, WAS patient platelets, though hypersusceptible to late-phase activation events such as phosphatidylserine exposure and microparticle release, 8 are nonetheless capable of normal early responses, including actin polymerization, shape change, and others. [8][9][10] To determine whether N-WASP might contribute to early cytoarchitectural reorganization events, we examined blood cells from healthy control subjects and from patients for the content of this protein. We also compared WASP and N-WASP for stability under conditions of platelet activation. Patients, materials, and methods PatientsWAS was diagnosed in patients, all previously described, 7 on the basis of male sex, thrombocytopenia with small platelets, eczema, immunodeficiency of variable clinical severity, family history (in some cases), and identific...

WAVE/Scars in platelets

“…19 However, a more recent report suggested that platelets express very small amounts of N-WASP. 20 These observations are consistent with those of Hartwig et al 13 who had provided evidence earlier that, for platelets in suspension, Rac rather than Cdc42 is essential for actin polymerization. However, both Rac and Cdc42 are reportedly involved in the development of lamellipodia in spreading platelets.…”

“…Consistent with the idea, only WAVE2 was found at linear structures (including the rim area) in lamellipodia following permeabilization and fixation of spread platelets ( Figure 5B and data not shown). On the basis of reports that platelets do not express N-WASP or at best very small amounts, 19,20 and that platelets from patients with WAS lacking WASP appear to spread normally on the surface, 17,18,45 we postulate that WAVEs (especially WAVE2) and cortactin rather than WASP or N-WASP play the predominant role in the regulation of actin polymerization in the lamellipodia of platelets. Recently, it was reported that WAVE2 requires phosphatidylinositol 3,4,5-trisphosphate for localization and promotion of lamellipodia in cell lines.…”

“…As WASP is a substrate for calpain in platelets, 16,20 we asked whether the same was true for WAVEs. When the platelets were treated with dibucaine, known to induce calpain activation in platelets, 16,33,34 we observed a significant loss of immunoreactive proteins that interacted with anti-WAVEs ( Figure 4A-C, lane 2).…”

“…The finding that the SH3 domain of IRSp53 binds strongly to WAVE2, weakly to WAVE1, and not at all to WAVE3 is in complete agreement with the results of the previous report that used cell lines. 20 Unlike WASP, neither WAVE1 nor WAVE2 binds to the amino terminal SH3 domain of CrkL, and WAVE3 does not appear to bind to any of these GST-fusion proteins. It has previously been reported that only WAVE1 among the WAVE proteins binds to protein kinase A.…”

WIP is a chaperone for Wiskott–Aldrich syndrome protein (WASP)

“…WASP in platelet lysates is degraded by the calcium-dependent protease calpain (16). We first determined whether WASP is a direct substrate for calpain.…”