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...

Tissue factor (TF) circulates in plasma, largely on monocyte/macrophage-derived microvesicles that can bind activated platelets through a mechanism involving P-selectin glycoprotein ligand-1 (PSGL-1) on the microvesicles and P-selectin on the platelets. We found these mi-crovesicles to be selectively enriched in both TF and PSGL-1, and deficient in CD45, suggesting that they arise from distinct membrane microdomains. We investigated the possibility that mi-crovesicles arise from cholesterol-rich lipid rafts and found that both TF and PSGL-1, but not CD45, localize to lipid rafts in blood monocytes and in the mono-cytic cell line THP-1. Consistent with a raft origin of TF-bearing microvesicles, their shedding was significantly reduced with depletion of membrane cholesterol. We also evaluated the interaction between TF-bearing microvesicles and plate-lets. Microvesicles bound only activated platelets, and required PSGL-1 to do so. The microvesicles not only bound the activated platelets, they fused with them, transferring both proteins and lipid to the platelet membrane. Fusion was blocked by either annexin V or an antibody to PSGL-1 and had an important functional consequence: increasing the proteolytic activity of the TF-VIIa complex. These findings suggest a mechanism by which all of the membrane-bound reactions of the coagulation system can be localized to the surface of activated platelets.

Fibrinogen (Fbg) mediates platelet aggregtion by its interaction with the platelet glycoprotein ilb-Mia (integrin as 3). Peptides containing the amino acid sequence RGD derived from the a chain (residues a95-97 and residues aS72-574) and the sequence HILGGAKQAGDV derived from the carboxyl terminus of the y chain of Fbg (residues vy400-411) inhibit these interactions.

These results indicate that P-selectin interaction with a ligand, different from PSGL-1 or GP Ib, stabilizes initial GP IIb/IIIa-fibrinogen interactions, allowing the formation of large stable platelet aggregates.

• Brain-derived cellular microparticles induce systemic coagulopathy in traumatic brain injury.• Platelets facilitate the transmigration of brain microparticles through the endothelial barrier into the circulation.Traumatic brain injury (TBI) is associated with coagulopathy, although it often lacks 2 key risk factors: severe bleeding and significant fluid resuscitation associated with hemorrhagic shock. The pathogenesis of TBI-associated coagulopathy remains poorly understood. We tested the hypothesis that brain-derived microparticles (BDMPs) released from an injured brain induce a hypercoagulable state that rapidly turns into consumptive coagulopathy. Here, we report that mice subjected to fluid percussion injury (1.9 6 0.1 atm) developed a BDMP-dependent hypercoagulable state, with peak levels of plasma glial cell and neuronal BDMPs reaching 17 496 6 4833/mL and 18 388 6 3657/mL 3 hours after TBI, respectively. Uninjured mice injected with BDMPs developed a dosedependent hyper-turned hypocoagulable state measured by a progressively prolonged clotting time, fibrinogen depletion, and microvascular fibrin deposition in multiple organs. The BDMPs were 50 to 300 nm with intact membranes, expressing neuronal or glial cell markers and procoagulant phosphatidylserine and tissue factor. Their procoagulant activity was greater than platelet microparticles and was dose-dependently blocked by lactadherin. Microparticles were produced from injured hippocampal cells, transmigrated through the disrupted endothelial barrier in a platelet-dependent manner, and activated platelets. These data define a novel mechanism of TBI-associated coagulopathy in mice, identify early predictive markers, and provide alternative therapeutic targets. (Blood. 2015;125(13):2151-2159

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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.

Key Points Mitochondria were released from traumatically injured brain into systemic circulation and exposed CL on their surface. CL-exposed mitochondria are highly procoagulant and induced traumatic brain injury–associated coagulopathy.