Swapan K. Dasgupta

Essential role for Nix in autophagic maturation of erythroid cells

Erythroid cells undergo enucleation and the removal of organelles during terminal differentiation. Although autophagy has been suggested to mediate the elimination of organelles for erythroid maturation, the molecular mechanisms underlying this process remain undefined. Here we report a role for a Bcl-2 family member, Nix (also called Bnip3L), 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 (ΔΨm), and treatment with uncoupling chemicals or a BH3 mimetic induced the loss of ΔΨm and restored the sequestration of mitochondria into autophagosomes in Nix-/- erythroid cells. These results suggest that Nix-dependent loss of ΔΨm 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.

Lactadherin and clearance of platelet-derived microvesicles

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 phosphatidylserine-expressing lymphocytes and red blood cells, in the clearance of platelet microvesicles. Platelet-derived microvesicles were labeled with BODIPY-maleimide 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. Lactadherin-deficient 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, lactadherin-deficient 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 platelet-derived microvesicles from the circulation and that a defective clearance can induce a hypercoagulable state.

Developmental Endothelial Locus-1 (Del-1) Mediates Clearance of Platelet Microparticles by the Endothelium

Background— Phosphatidylserine-expressing microparticles circulate in blood with a short half-life of <10 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 (P=0.027) and by annexin A5 (P=0.027), abciximab (P=0.027), a monoclonal antibody to integrin &agr;V&bgr;3 (P=0.027), and chlorpromazine (P=0.027). 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 (3.07±1.9 versus 1.09±1.3, P=0.02) and liver (2.85±1.1 versus 1.35±0.92, P=0.01). Furthermore, after endotoxin administration, Del-1–deficient mice displayed an increase in the level of microparticles compared with wild-type mice (P=0.02). Conclusions— These studies show a physiological role for Del-1 in the clearance of phosphatidylserine-expressing microparticles by endothelium.

A remarkable bismuth nitrate-catalyzed protection of carbonyl compounds

Abstract Bismuth nitrate has been found to be an outstanding catalyst for the protection of carbonyl compounds as acetal, ketal, mixed ketal and thioketal with an excellent yield.

Platelet senescence and phosphatidylserine exposure

BACKGROUND: The exposure of phosphatidylserine occurs during platelet (PLT) activation and during in vitro storage. Phosphatidylserine exposure also occurs during apoptosis after the release of mitochondrial cytochrome c. We have examined the role of cytochrome c release, mitochondrial membrane potential (ΔΨm), and cyclophilin D (CypD) in phosphatidylserine exposure due to activation and storage.

Role of lactadherin in the clearance of phosphatidylserine-expressing red blood cells.

BACKGROUND: In red blood cells (RBCs) anionic phospholipids, such as phosphatidylserine, are present in the inner leaflet of the membrane bilayer. Exposure of phosphatidylserine occurs during senescence and during long‐term storage of RBCs and is considered as the tag for removal from the circulation by macrophages. Lactadherin is a phosphatidylserine‐binding glycoprotein secreted by macrophages that promotes the engulfment of phosphatidylserine‐expressing apoptotic lymphocytes. This study investigates the role of lactadherin in the phagocytosis of phosphatidylserine‐expressing RBCs.

A new entry to N-unsubstituted β-lactams through a solid-phase approach

A remarkable new entry to N-unsubstituted β-lactams using rink resin as the solid-support has been developed.

Antiplatelet factor 4/heparin antibodies in patients with gram negative bacteremia.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated syndrome of thrombocytopenia and prothrombotic state that follows exposure to heparin. However, spontaneous HIT has been described in the setting of infection, without evidence of previous heparin administration. Since PF4 binds to lipid A portion of lipopolysaccharide, we tested for the presence of antiPF4/heparin antibodies in patients with gram-negative bacteremia. Patients with bacteremia had higher titers of antiPF4/heparin antibodies compared to normal controls 26.3 ± SD 34 units, N=32 versus 6.3 ± SD 2.38 units, N=10, P=0.001. FITC-labeled PF4 interacted with lipopolysaccharide in a concentration-dependent manner as determined by quenching of the emission spectrum following excitation at λ 488. In addition, immunoaffinity purified antiPF4/Heparin antibodies from 3 patients with HIT cross-reacted with PF4/heparin complex. These results show that PF4/LPS complex is immunogenic and can elicit cross-reacting antibodies against PF4/Heparin, providing an explanation for the presence of these antibodies in individuals, who were never been exposed to heparin before. These antibodies may also be at least partly responsible for the thrombocytopenia associated with infection.

Dasatinib inhibits actin fiber reorganization and promotes endothelial cell permeability through RhoA-ROCK pathway

Treatment with dasatinib, a tyrosine kinase inhibitor, is associated with edema, pleural effusion, and pulmonary edema. We investigated the effect of dasatinib on the barrier function of human microvascular endothelial cells‐1 (HMEC‐1) in vitro and in vivo. The permeability of HMEC‐1 to fluorescein isothiocyante (FITC)‐dextran increased in Transwell chambers within 5 min following the addition of therapeutic concentrations of dasatinib. The change in permeability was associated with increased activation of RhoA GTPase and its effector Rho‐associated coiled‐coil kinase 1(ROCK1). RhoA inhibitor C3 transferase almost completely inhibited dasatinib‐induced increase in permeability. Under similar conditions, imatinib had no effect on permeability or activation of RhoA. Since integrin‐induced cell spreading suppresses RhoA activation, we examined the effect of dasatinib on cell spreading on fibronectin substrate. Dasatinib impaired endothelial cell spreading in a concentration‐dependent manner and induced disorganization of actin fibers. Tyrosine kinases play an essential role in transmitting signals from integrins to RhoA and we examined tyrosine phosphorylation of several cytoskeletal proteins. Dasatinib markedly inhibited tyrosine phosphorylation of p130 Crk‐associated substrate (p130cas), paxillin and vinculin. These results suggest that the inhibition of tyrosine phosphorylation of the focal adhesion plaque components by dasatinib may alter the assembly of actin fibers resulting in the activation of RhoA/ROCK pathway. Consistent with these findings, dasatinib‐induced increase in the permeability was blocked by ROCK inhibitor y27632. In vivo administration of y27632, significantly inhibited the dasatinib‐induced extravasation of Evans blue in mice and dasatinib‐induced increase in microvascular permeability was attenuated in ROCK1‐deficient mice. These findings suggest that ROCK inhibitors could serve as therapeutic modalities to ameliorate the dasatinib‐induced pulmonary changes.

Rho Associated Coiled-Coil Kinase-1 Regulates Collagen-Induced Phosphatidylserine Exposure in Platelets

Background The transbilayer movement of phosphatidylserine mediates the platelet procoagulant activity during collagen stimulation. The Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 inhibits senescence induced but not activation induced phosphatidylserine exposure. To investigate further the specific mechanisms, we now utilized mice with genetic deletion of the ROCK1 isoform. Methods and Results ROCK1-deficient mouse platelets expose significantly more phosphatidylserine and generate more thrombin upon activation with collagen compared to wild-type platelets. There were no significant defects in platelet shape change, aggregation, or calcium response compared to wild-type platelets. Collagen-stimulated ROCK1-deficient platelets also displayed decreased phosphorylation levels of Lim Kinase-1 and cofilin-1. However, there was no reduction in phosphorylation levels of myosin phosphatase subunit-1 (MYPT1) or myosin light chain (MLC). In an in vivo light/dye-induced endothelial injury/thrombosis model, ROCK1-deficient mice presented a shorter occlusion time in cremasteric venules when compared to wild-type littermates (3.16 ± 1.33 min versus 6.6 ± 2.6 min; p = 0.01). Conclusions These studies define ROCK1 as a new regulator for collagen-induced phosphatidylserine exposure in platelets with functional consequences on thrombosis. This effect was downstream of calcium signaling and was mediated by Lim Kinase-1 / cofilin-1-induced cytoskeletal changes.