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 ␥-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 Ϫ1 ) or high (1500 s

The platelet collagen receptor glycoprotein (GP) VI-Fc receptor ␥-chain (FcR␥) complex transduces signals in an immunoreceptorlike manner. We examined a role for the Triton X-100-insoluble membrane rafts in GPVI-FcR␥ complex signaling. Methyl-␤-cyclodextrin (M␤CD)-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-FcR␥ complex. The GPVI-FcR␥ 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 M␤CD treatment. Thus, we provide the first evidence that the GPVI-FcR␥ complex is constitutively and functionally associated with membrane rafts. IntroductionPlasma membranes of many cell types contain microdomains commonly referred to as membrane or lipid rafts. They are enriched in sphingolipid and cholesterol that would preferentially self-associate to form lateral lipid assemblies in an unsaturated glycerophospholipid environment. 1,2 Membrane rafts are resistant to solubilization in nonionic detergent but can be isolated from low-density fractions after flotation in a sucrose gradient. A variety of specific proteins are concentrated in raft domains, including many glycophosphatidylinositol-anchored proteins, Src family kinases, and linker for activation of T cells (LAT). Evidence has accumulated for membrane raft-dependent scaffolding of signaling complexes, mostly shown by studies on immunoreceptor signaling including T-and B-cell antigen receptors and Fc⑀ receptor I. 3,4 The functional significance of membrane rafts has been strengthened by the finding that receptor aggregates in membrane rafts can signal in a ligandindependent manner. 5 Dorahy et al 6,7 first identified the Triton X-100-insoluble membrane rafts of resting platelets that are enriched in Src, Lyn, and CD36. However, the exact role for membrane rafts of platelets remains to be clarified.Platelets adhere to the extracellular matrix protein, collagen, at the site of vascular damage and become activated through specific membrane receptors, resulting in shape change, granule release, and aggregation. Previous studies have identified the integrin ␣ 2 ␤ 1 and the glycoprotein (GP) VI-Fc receptor ␥-chain (FcR␥) complex as 2 major receptors involved in plateletcollagen interaction. 8 It has been well established that the GPVI-FcR␥ complex signals in an immunoreceptorlike manner; the complex engagement causes activation of the Src family kinases, including Fyn, which then phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAMs) of FcR␥. Syk kinase binds the tyrosine-phosphorylated ITAMs through its SH2 domain. It is then activated and phosphorylates phospholipase C␥2 (PLC␥2), leading to its activation. The signals further expand through tyrosine phosphorylation of LAT, SLP-76, and others. 8 Clemetson et al 9 first reported the cloning of GPVI, a member of the imm...

CCAAT/enhancer-binding protein alpha (CEBPA) mutations are a favorable prognostic factor in adult acute myeloid leukemia (AML) patients; however, few studies have examined their significance in pediatric AML patients. Here we examined the CEBPA mutation status and clinical outcomes of pediatric AML patients treated in the AML-05 study. We found that 47 (14.9%) of the 315 evaluable patients harbored mutations in CEBPA; 26 cases (8.3%) harbored a single mutation (CEBPA-single) and 21 (6.7%) harbored double or triple mutations (CEBPA-double). After excluding core-binding factor-AML cases, patients harboring CEBPA mutations showed better overall survival (OS; P=0.048), but not event-free survival (EFS; P=0.051), than wild-type patients. Multivariate analysis identified CEBPA-single and CEBPA-double as independent favorable prognostic factors for EFS in the total cohort (hazard ratio (HR): 0.47 and 0.33; P=0.02 and 0.01, respectively). CEBPA-double was also an independent favorable prognostic factor for OS (HR: 0.30; P=0.04). CEBPA-double remained an independent favorable factor for EFS (HR: 0.28; P=0.04) in the normal karyotype cohort. These results suggest that CEBPA mutations, particularly CEBPA-double, are an independent favorable prognostic factor in pediatric AML patients, which will have important implications for risk-stratified therapy.