Erythroid cells undergo enucleation and the removal of organelles during terminal differentiation 1-3 . Although autophagy has been suggested to mediate the elimination of organelles for erythroid maturation 2-6 , the molecular mechanisms underlying this process remain undefined. Here we report a role for a Bcl-2 family member, Nix (also called Bnip3L) 7-9 , in the regulation of erythroid maturation through mitochondrial autophagy. Nix −/− mice developed anaemia with reduced mature erythrocytes and compensatory expansion of erythroid precursors. Erythrocytes in the peripheral blood of Nix −/− mice exhibited mitochondrial retention and reduced lifespan in vivo. Although the clearance of ribosomes proceeded normally in the absence of Nix, the entry of mitochondria into autophagosomes for clearance was defective. Deficiency in Nix inhibited the loss of mitochondrial membrane potential (ΔΨ m ), and treatment with uncoupling chemicals or a BH3 mimetic induced the loss of ΔΨ m and restored the sequestration of mitochondria into autophagosomes in Nix −/− erythroid cells. These results suggest that Nix-dependent loss of ΔΨ m is important for targeting the mitochondria into autophagosomes for clearance during erythroid maturation, and interference with this function impairs erythroid maturation and results in anaemia. Our study may also provide insights into molecular mechanisms underlying mitochondrial quality control involving mitochondrial autophagy.Nix, a BH3-only member of the Bcl-2 family, is upregulated in erythroid cells undergoing terminal differentiation 10 . To determine the potential function for Nix in erythroid maturation, we generated Nix −/− mice using embryonic stem (ES) cells with a gene trap insertion between exons 3 and 4 of Nix ( Supplementary Fig. 2). We first examined red blood cells in the peripheral blood (RBCs), including reticulocytes and erythrocytes, in Nix −/− mice. Although RBC counts were decreased (Supplementary Table 1), polychromasia and increased reticulocytes were observed in Nix −/− mice ( Fig. 1a and Supplementary Fig. 3a). We also examined RBCs for the expression of an erythroid cell marker, glycophorin-A-associated Ter119, and for transferrin receptor CD71, which is downregulated during terminal erythroid differentiation 11,12 . Although Ter119 low CD71 high and Ter119 + CD71 high early erythroblasts 13 were absent in the peripheral blood, a significant increase in Ter119 + CD71 + reticulocytes was observed in Nix −/− mice (Fig. 1b). Electron microscopy also showed more irregularly shaped cellsCorrespondence and requests for materials should be addressed to M.C. (minc@bcm.tmc.edu) or J.W. (jinwang@bcm.tmc.edu). Author Contributions H.S. conducted the majority of the experiments, supervised by J.W. and M.C.; P.T. stained spleen sections and blood smears; S.K.D. measured osmotic fragility and assisted with biotin and CMFDA labelling; A.S. performed RT-PCR for Epo; J.T.P. and P.T. provided experimental advice; M.C. and J.W. generated the Nix −/− mice, designed experiments and...

Background-Phosphatidylserine-expressing microparticles circulate in blood with a short half-life of Ͻ10 minutes. We tested the role of an endothelium-derived phosphatidylserine-binding opsonin, developmental endothelial locus-1 (Del-1), in the uptake of platelet microparticles. Methods and Results-Cultured human umbilical vein and microvascular endothelial cells avidly engulf BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-maleimide-labeled platelet microparticles. Microparticle uptake was inhibited by a monoclonal antibody to Del-1 (Pϭ0.027) and by annexin A5 (Pϭ0.027), abciximab (Pϭ0.027), a monoclonal antibody to integrin ␣V␤3 (Pϭ0.027), and chlorpromazine (Pϭ0.027). These results suggest that Del-1 mediates phosphatidylserine-and integrin-dependent endothelial uptake of microparticles by endocytosis. To assess the in vivo significance, we infused fluorescent platelet microparticles into the inferior vena cava of mice and harvested endothelial cells from the pulmonary and systemic circulation. Compared with their wild-type littermates, Del-1-deficient mice had decreased uptake in endothelial cells in lung (3.07Ϯ1.9 versus 1.09Ϯ1.

The transbilayer movement of phosphatidylserine from the inner to the outer leaflet of the membrane bilayer during platelet activation is associated with the release of procoagulant phosphatidylserine-rich small membrane vesicles called platelet-derived microvesicles. We tested the effect of lactadherin, which promotes the phagocytosis of phosphatidylserineexpressing lymphocytes and red blood cells, in the clearance of platelet microvesicles. Platelet-derived microvesicles were labeled with BODIPYmaleimide and incubated with THP-1-derived macrophages. The extent of phagocytosis was quantified by flow cytometry. Lactadherin promoted phagocytosis in a concentration-dependent manner with a half-maximal effect at approximately 5 ng/mL. Lactadherindeficient mice had increased number of platelet-derived microvesicles in their plasma compared with their wild-type littermates (950 ؎ 165 vs 4760 ؎ 650; P ‫؍‬ .02) and generated 2-fold more thrombin. In addition, splenic macrophages from lactadherin-deficient mice showed decreased capacity to phagocytose platelet-derived microvesicles. In an in vivo model of light/dye-induced endothelial injury/thrombosis in the cremasteric venules, lactadherindeficient mice had significantly shorter time for occlusion compared with their wild-type littermate controls (5.93 ؎ 0.43 minutes vs 9.80 ؎ 1.14 minutes;P ‫؍‬ .01). These studies show that lactadherin mediates the clearance of phosphatidylserine-expressing plateletderived microvesicles from the circulation and that a defective clearance can induce a hypercoagulable state. (Blood.

Endotoxemia promotes adhesive interactions between platelets and microvascular endothelium in vivo. We sought to determine whether endotoxin (lipopolysaccharide, LPS) modified platelet thrombus formation in mouse cremaster venules and whether Toll-like receptor 4 (TLR4) and neutrophils were involved in the response. Intravital videomicroscopy was performed in the cremaster microcirculation of pentobarbital-anesthetized mice; venular platelet thrombi were induced with a light/dye endothelial injury model. C57BL/6 mice treated with Escherichia coli endotoxin had enhanced rates of venular platelet thrombus formation: the time to microvessel occlusion was reduced by approximately 50% (P < 0.005) compared with saline-treated animals. Enhanced microvascular thrombosis was evident as early as 2 h after LPS administration. LPS had no effect on thrombosis in either of two mouse strains with altered TLR4 signaling (C57BL/10ScNJ or C3H/HeJ), whereas it enhanced thrombosis in the control strains (C57BL/10J and C3H/HeN). LPS also enhanced platelet adhesion to endothelium in the absence of light/dye injury. Platelet adhesion, but not enhanced thrombosis, was inhibited by depletion of circulating neutrophils. LPS failed to enhance platelet aggregation ex vivo and did not influence platelet P-selectin expression, a marker of platelet activation. These findings support the notion that endotoxemia promotes platelet thrombus formation independent of neutrophils and without enhancement of platelet aggregation, via a TLR4-dependent mechanism.

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