Trapping of CDC42 C-terminal variants in the Golgi drives pyrin inflammasome hyperactivation.

Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Effectiveness of IL-1ß-blocking therapy indicates that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42R186C, we found that patient-derived cells secreted larger amounts of IL-1ß in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42R186C protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42*192C*24 that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but not that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave the way for elucidating the mechanism of pyrin inflammasome formation.

Acute myeloid leukaemia with myelodysplastic features in children: a report of Japanese Paediatric Leukaemia/Lymphoma Study Group.

The clinical characteristics and prognostic relevance of acute myeloid leukaemia (AML) with myelodysplastic features remains to be clarified in children. We prospectively examined 443 newly diagnosed patients in a multicentre clinical trial for paediatric de novo AML, and found 'AML with myelodysplasia-related changes' (AML-MRC) according to the 2008 World Health Organization classification in 93 (21·0%), in whom 59 were diagnosed from myelodysplasia-related cytogenetics alone, 28 from multilineage dysplasia alone and six from a combination of both. Compared with 111 patients with 'AML, not otherwise specified' (AML-NOS), patients with 'AML-MRC' presented at a younger age, with a lower white blood cell count, higher incidence of 20-30% bone marrow blasts, unfavourable cytogenetics and a lower frequency of Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), NPM1 and CEBPA mutations. Complete remission rate and 3-year probability of event-free survival were significantly worse in 'AML-MRC' patients (67·7 vs. 85·6%, P < 0·01, 37·1% vs. 53·8%, P = 0·02, respectively), but 3-year overall survival and relapse-free survival were comparable with 'AML-NOS' patients. By multivariate analysis, FLT3-ITD was solely associated with worse overall survival. These results support the distinctive features of the category 'AML-MRC' even in children.

Intracellular localization of glycoprotein VI in human platelets and its surface expression upon activation.

Glycoprotein (GP) VI is a major receptor for collagen and belongs to the immunoglobulin super family. Here, we examined the localization of GPVI in resting and activated human platelets by immunogold scanning and transmission electron microscopy and flow cytometry. Ultrastructural observation detected immunolabelling for GPVI that was distributed uniformly over the entire surface of resting platelets, and revealed that GPVI was also localized on both the membranes of the surface-connected open canalicular system (OCS) and alpha-granules. The OCS- and alpha-granule-associated GPVI pools were an estimated 35.4 +/- 3.2% (mean +/- standard deviation) of the total. Little GPVI labelling was observed in any part of GPVI-deficient platelets. A remarkable time-dependent increase in GPVI surface expression was found by flow cytometry when platelets were activated by collagen-related peptide (CRP) and convulxin. The GPVI-mediated activation of platelets by CRP or convulxin resulted in similar ultrastructural changes and an increased GPVI labelling density on the activated platelet surface, which was accompanied by a decreased interior expression. GPVI was also expressed on microparticles generated from activated platelets. Thus, our study demonstrates that platelets have internal pools of GPVI, and that GPVI is increasingly redistributed to the surface membrane and to microparticles during platelet activation.

Involvement of glycoprotein VI in platelet thrombus formation on both collagen and von Willebrand factor surfaces under flow conditions.

BACKGROUND: We studied the role of glycoprotein (GP) VI in platelet adhesion and thrombus formation on the immobilized collagen and von Willebrand factor (vWF) surface under flow conditions. METHODS AND RESULTS: Whole blood obtained from 2 patients with GP VI-deficient platelets and the effects of the Fab of anti-GP VI antibody (Fab/anti-GP VI) were tested. Blood containing platelets rendered fluorescent by mepacrine was perfused on immobilized type I collagen or vWF under controlled wall shear rate. Platelet adhesion and thrombus formation were detected by epifluorescent videomicroscopy. The percentage of surface coverage by the platelets was calculated. Fc receptor gamma-chain and spleen tyrosine kinase (Syk) were immunoprecipitated from the lysate of platelets stimulated by vWF plus ristocetin and then analyzed by antiphosphotyrosine immunoblotting. No platelet attachment was seen on the surface of collagen even after 9 minutes of perfusion of blood at relatively low (100 s(-1)) or high (1500 s(-1)) wall shear rate, either in the case of blood containing GP VI-deficient platelets or in the presence of Fab/anti-GP VI, whereas significant platelet thrombus formation was noted after control blood perfusion. Such interference with the actions of GP VI also reduced firm platelet adhesion on immobilized vWF. vWF-induced tyrosine phosphorylation of GP VI-associated Fc receptor gamma-chain followed by Syk activation occurred in normal platelets, but little activation of Syk occurred in GP VI-deficient platelets. CONCLUSIONS: GP VI plays crucial roles in platelet thrombus formation on the surface of collagen under flow conditions in humans and is also involved in the process of firm platelet adhesion on the surface of vWF.

Constitutive and functional association of the platelet collagen receptor glycoprotein VI-Fc receptor gamma-chain complex with membrane rafts.

The platelet collagen receptor glycoprotein (GP) VI-Fc receptor gamma-chain (FcRgamma) complex transduces signals in an immunoreceptorlike manner. We examined a role for the Triton X-100-insoluble membrane rafts in GPVI-FcRgamma complex signaling. Methyl-beta-cyclodextrin (MbetaCD)-induced disruption of the membrane rafts inhibited not only platelet aggregation and secretion but also tyrosine phosphorylation of signaling molecules on stimulation through the GPVI-FcRgamma complex. The GPVI-FcRgamma complex was constitutively associated with membrane rafts wherein the Src family kinases and LAT were also present. Their association was not affected by the complex engagement but was highly sensitive to MbetaCD treatment. Thus, we provide the first evidence that the GPVI-FcRgamma complex is constitutively and functionally associated with membrane rafts.