Erythrophagocytosis in autoimmune immunoglobulin A-mediated hemolysis.

INTRODUCTION: Warm antibody-mediated autoimmune hemolysis (WAIHA) is most often due to immunoglobulin G (IgG) antibodies and is detected by direct antiglobulin test (DAT). However, about 10% cases of hemolytic anemia are DAT negative. Herein, we describe a patient with DAT-negative hemolytic anemia due to an anti-IgA antibody. We investigate if isolated IgA can promote erythrophagocytosis. METHODS: We isolated patient and control IgA on Jacalin agarose. Isolated IgA was used to sensitize healthy ABO/Rh-matched donor red blood cells (RBC). Antibody binding was examined by flowcytometry. The effect of IgA on erythrophagocytosis was evaluated using Macrophage colony stimulating factor 1 (M-CSF)-differentiated autologous macrophages by Giemsa staining and immunofluorescence microscopy. RESULTS: We show that isolated IgA from the serum can bind to red cells. In addition, RBCs were phagocytosed efficiently by autologous macrophages in the presence of patient IgA. CONCLUSION: Our results show that IgA antibodies are capable of inducing erythrophagocytosis like IgG antibodies in the absence of complement fixation.

Lactadherin promotes microvesicle clearance to prevent coagulopathy and improves survival of severe TBI mice.

Coagulopathy is common in patients with traumatic brain injury (TBI) and predicts poor clinical outcomes. We have shown that brain-derived extracellular microvesicles, including extracellular mitochondria, play a key role in the development of TBI-induced coagulopathy. Here, we further show in mouse models that the apoptotic cell-scavenging factor lactadherin, given at a single dose of 400 µg/kg 30 minutes before (preconditioning) or 30 minutes after cerebral fluid percussion injury, prevented coagulopathy as defined by clotting time, fibrinolysis, intravascular fibrin deposition, and microvascular bleeding of the lungs. Lactadherin also reduced cerebral edema, improved neurological function, and increased survival. It achieved these protective effects by enhancing the clearance of circulating microvesicles through phosphatidylserine-mediated phagocytosis. Together, these results identify the scavenging system for apoptotic cells as a potential therapeutic target to prevent TBI-induced coagulopathy and improve the outcome of TBI.

von Willebrand factor enhances microvesicle-induced vascular leakage and coagulopathy in mice with traumatic brain injury.

von Willebrand factor (VWF) is an adhesive ligand, and its activity is proteolytically regulated by the metalloprotease ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat 13). An elevated level of plasma VWF has been widely considered a marker for endothelial cell activation in trauma and inflammation, but its causal role in these pathological conditions remains poorly defined. Using a fluid percussion injury mouse model, we demonstrated that VWF released during acute traumatic brain injury (TBI) was activated and became microvesicle-bound. The VWF-bound microvesicles promoted vascular leakage and systemic coagulation. Recombinant ADAMTS-13 given either before or after TBI reduced the VWF reactivity with minimal influence on VWF secretion. rADAMTS-13 protected the integrity of endothelial cell barriers and prevented TBI-induced coagulopathy by enhancing VWF cleavage without impairing basal hemostasis. Promoting microvesicle clearance by lactadherin had efficacy similar to that of rADAMTS-13. This study uncovers a novel synergistic action between VWF and cellular microvesicles in TBI-induced vascular leakage and coagulopathy and demonstrates protective effects of rADAMTS-13.

Extracellular mitochondria released from traumatized brains induced platelet procoagulant activity.

Coagulopathy often develops soon after acute traumatic brain injury and its cause remains poorly understood. We have shown that injured brains release cellular microvesicles that disrupt the endothelial barrier and induce consumptive coagulopathy. Morphologically intact extracellular mitochondria accounted for 55.2% of these microvesicles, leading to the hypothesis that these extracellular mitochondria are metabolically active and serve as a source of oxidative stress that activates platelets and renders them procoagulant. In testing this hypothesis experimentally, we found that the extracellular mitochondria purified from brain trauma mice and those released from brains subjected to freeze-thaw injury remained metabolically active and produced reactive oxygen species. These extracellular mitochondria bound platelets through the phospholipid-CD36 interaction and induced α-granule secretion, microvesiculation, and procoagulant activity in an oxidant-dependent manner, but failed to induce aggregation. These results define an extracellular mitochondria-induced and redox-dependent intermediate phenotype of platelets that contribute to the pathogenesis of traumatic brain injury-induced coagulopathy and inflammation.

Cofilin-1-induced actin reorganization in stored platelets.

BACKGROUND: During platelet storage, there are extensive changes in cytoskeleton and phosphatidylserine exposure. The intrinsic mitochondrial pathway of apoptosis, activated in stored platelets, is a major mediator these changes. Cofilin-1 is an effector of actin reorganization. We examined the effect of cofilin-1 deficiency on cytoskeleton and phosphatidylserine exposure during storage and following activation of apoptosis. METHODS AND RESULTS: We assessed actin filaments by Alexa-647-phalloidin and phosphatidylserine exposure by fluorescein isothiocyanate-lactadherin by fluorescence microscopy. In fresh platelets, actin filaments are distributed in the subcortical region, and they do not express phosphatidylserine in the outer surface. In stored platelets, there is retraction of actin filaments from the subcortical region with increased phosphatidylserine expression. These changes are seen in 20% of platelets of 6 days old and increases further with storage. Treatment with ABT-737, which activates the mitochondrial apoptosis, induces similar cytoskeletal changes in actin filaments with increased phosphatidylserine. Cofilin-1 is activated in stored platelets as well as in ABT-737 treated platelets by dephosphorylation. In cofilin-1 deficient murine platelets actin filaments are abnormal and ABT-737 induces less phosphatidylserine. Despite these changes in vitro, platelet survival of cofilin-1 deficient platelets in mice was not significantly different from their wild-type controls. CONCLUSION: These results show that cofilin-1 plays a role in apoptosis-induced actin rearrangement and phosphatidylserine exposure during storage. Despite the defects in platelet cytoskeleton and phosphatidylserine exposure in cofilin-1-deficient platelets, the in vivo life span of platelets is similar to littermate controls, indicating multiple redundant pathways for the clearance of platelets in vivo.

Cardiolipin-mediated procoagulant activity of mitochondria contributes to traumatic brain injury-associated coagulopathy in mice.

Cardiolipin (CL) is an anionic phospholipid located exclusively in the mitochondrial inner membrane. Its presence in blood indicates mitochondrial damage and release from injured cells. Here, we report the detection of CL-exposed brain-derived mitochondrial microparticles (mtMPs) at 17 547 ± 2677/µL in the peripheral blood of mice subjected to fluid percussion injury to the brain. These mtMPs accounted for 55.2% ± 12.6% of all plasma annexin V-binding microparticles found in the acute phase of injury. They were also released from cultured neuronal and glial cells undergoing apoptosis. The mtMPs synergized with platelets to facilitate vascular leakage by disrupting the endothelial barrier. The disrupted endothelial barrier allowed the release of mtMPs into the systemic circulation to promote coagulation in both traumatically injured and mtMP- or CL-injected mice, leading to enhanced fibrinolysis, vascular fibrin deposition, and thrombosis. This mtMP-induced coagulation was mediated by CL transported from the inner to the outer mitochondrial membrane and was blocked by the scavenging molecule lactadherin. The mtMP-bound CL was ∼1600 times as active as purified CL in promoting coagulation. This study uncovered a novel procoagulant activity of CL and CL-exposed mitochondria that may contribute to traumatic brain injury-associated coagulopathy and identified potential pathways to block this activity.

Brain-derived microparticles induce systemic coagulation in a murine model of traumatic brain injury.

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 ± 0.1 atm) developed a BDMP-dependent hypercoagulable state, with peak levels of plasma glial cell and neuronal BDMPs reaching 17 496 ± 4833/µL and 18 388 ± 3657/µL 3 hours after TBI, respectively. Uninjured mice injected with BDMPs developed a dose-dependent 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.

A small amount of cyclooxygenase 2 (COX2) is constitutively expressed in platelets.

Cyclooxygenase (COX) is the rate-limiting enzyme in conversion of arachidonic acid to prostanoids, and has two isoforms, COX1 and COX2, which share ~65% amino acid homology. COX1 is universally expressed in many cell types including platelets; however, expression of COX2 is known to be more limited. We examined expression of COX2 mRNA and protein in platelets and platelet-derived microparticles (MPs); using quantitative RT-PCR, immunostaining, and Western blotting. We have detected a significant amount of COX2 in platelets, both at mRNA and protein levels. We found that COX1/COX2 mRNA and protein ratios in platelets were 370:1 and 17:1, respectively. Expression level of COX2 in platelets was less than COX1, but comparable to the expression of COX2 in malignant epithelial cells. Considering the important role of COX2 in tumorigenesis and thrombosis, and the large number of circulating platelets, we propose that platelet COX2 may play an important role in physiologic and pathologic conditions.

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 (DeltaPsi(m)), and treatment with uncoupling chemicals or a BH3 mimetic induced the loss of DeltaPsi(m) and restored the sequestration of mitochondria into autophagosomes in Nix(-/-) erythroid cells. These results suggest that Nix-dependent loss of DeltaPsi(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.

ß2-glycoprotein I promotes the clearance of circulating mitochondria.

ß2-glycoprotein I (ß2-Gp1) is a cardiolipin-binding plasma glycoprotein. It is evolutionarily conserved from invertebrates, and cardiolipin-bound ß2-Gp1 is a major target of antiphospholipid antibodies seen in autoimmune disorders. Cardiolipin is almost exclusively present in mitochondria, and mitochondria are present in circulating blood. We show that ß2-Gp1 binds to cell-free mitochondria (CFM) in the circulation and promotes its phagocytosis by macrophages at physiological plasma concentrations. Exogenous CFM had a short circulation time of less than 10 minutes in mice. Following infusion of CFM, ß2-Gp1-deficient mice had significantly higher levels of transfused mitochondria at 5 minutes (9.9 ± 6.4 pg/ml versus 4.0 ± 2.3 pg/ml in wildtype, p = 0.01) and at 10 minutes (3.0 ± 3.6 pg/ml versus 1.0 ± 0.06 pg/ml in wild-type, p = 0.033, n = 10). In addition, the splenic macrophages had less phagocytosed CFM in ß2-Gp1-deficient mice (24.4 ± 2.72% versus 35.6 ± 3.5 in wild-type, p = 0.001, n = 5). A patient with abnormal ß2-Gp1, unable to bind cardiolipin, has increased CFM in blood (5.09 pg/ml versus 1.26 ± 1.35 in normal) and his plasma induced less phagocytosis of CFM by macrophages (47.3 ± 1.6% versus 54.3 ± 1.3, p = 0.01) compared to normal plasma. These results show the evolutionarily conserved ß2-Gp1 is one of the mediators of the clearance of CFM in circulation.

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 αVß3 (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.

The activity of the androgen receptor variant AR-V7 is regulated by FOXO1 in a PTEN-PI3K-AKT-dependent way.

BACKGROUND: The androgen receptor (AR) AR-V7 splice isoform is a constitutively active outlaw transcription factor. Transition of prostate cancer (PC) to the castration-resistant phenotype correlates with AR-V7 accumulation, suggesting that PC progression in patients refractory to conventional therapy is due to the activity of this AR isoform. The mechanism of AR-V7 constitutive activation is not known. METHODS: We analyzed potential signaling pathways associated with AR-V7 constitutive activation in PTEN (-) PC-3 and LNCaP cells. We used transient and stable transfection, reporter gene assay, RNAi technology together with a number of kinase inhibitors to determine if AR-V7 activation is linked to a kinase-dependent signaling pathway. RESULTS: In these cell lines, AR-V7 transcriptional activity was inhibited by LY294002, Wortmanin, and AKT inhibitor II. Analysis of the contributing mechanisms demonstrated the involvement of the Phosphatidylinositol 3-kinase (PI3K)-AKT-FOXO1 signaling pathway, and a significant reduction of AR-V7 constitutive activity under conditions of PTEN reactivation. CONCLUSIONS: Our study identifies a pathway regulating AR-V7 constitutive activity and potential therapeutic targets for the treatment of castration-resistant PC.

Downregulated KLF2 in polycythemia vera and essential thrombocythemia induces prothrombotic gene expression.

Thromboses are major causes of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET) diseases associated with JAK2V617F mutation. However, the molecular mechanism(s) of increased thrombosis in PV and ET remain unknown. Kruppel-like factor 2 (KLF2) is a transcription factor that regulates expression of genes associated with inflammation and thrombosis; the absence of KLF2 in neutrophils causes thrombosis by inducing tissue factor. We studied the role of KLF2 in regulating prothrombotic gene expression in PV and ET. Neutrophils and platelets KLF2 expression in PV and ET was lower than the controls. Furthermore, in patients with thromboses, KLF2 transcripts were lower in platelets than those without thromboses. JAK2V617F allelic burden was inversely correlated with KLF2 transcript levels, suggesting JAK-STAT pathway may downregulate KLF2 expression. Whole transcriptome analyses of neutrophils and platelets showed that a lower KLF2 expression was associated with an upregulation of KLF2-regulated thrombotic genes. In addition, low KLF2 expression in platelets positively correlated with thrombotic events. In patients with PV and ET, KLF2 expression was induced by pegylated interferon alfa (PegINF-α) but not by hydroxyurea treatments. These data suggest that KLF2 may be a regulator of PV and ET thrombosis and a novel therapeutic target to prevent thrombosis.

Mutations in Hcfc1 and Ronin result in an inborn error of cobalamin metabolism and ribosomopathy.

Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.

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.

How Do Red Blood Cells Die?

Normal human red blood cells have an average life span of about 120 days in the circulation after which they are engulfed by macrophages. This is an extremely efficient process as macrophages phagocytose about 5 million erythrocytes every second without any significant release of hemoglobin in the circulation. Despite large number of investigations, the precise molecular mechanism by which macrophages recognize senescent red blood cells for clearance remains elusive. Red cells undergo several physicochemical changes as they age in the circulation. Several of these changes have been proposed as a recognition tag for macrophages. Most prevalent hypotheses for red cell clearance mechanism(s) are expression of neoantigens on red cell surface, exposure phosphatidylserine and decreased deformability. While there is some correlation between these changes with aging their causal role for red cell clearance has not been established. Despite plethora of investigations, we still have incomplete understanding of the molecular details of red cell clearance. In this review, we have reviewed the recent data on clearance of senescent red cells. We anticipate recent progresses in in vivo red cell labeling and the explosion of modern proteomic techniques will, in near future, facilitate our understanding of red cell senescence and their destruction.

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. STUDY DESIGN AND METHODS: The exposure of phosphatidylserine and the loss of ΔΨm were determined in a flow cytometer using fluorescein isothiocyanate-lactadherin and JC-1, a lipophilic cationic reporter dye. The role of CypD was determined with cyclosporin A and CypD-deficient murine PLTs. Cytochrome c-induced caspase-3 and Rho-associated kinase I (ROCK1) activation were determined by immunoblotting and using their inhibitors. RESULTS: Collagen- and thrombin-induced exposure of phosphatidylserine was accompanied by a decrease in ΔΨm. Cyclosporin A inhibited the phosphatidylserine exposure and the loss of ΔΨm. CypD(-/-) mice had decreased loss of ΔΨm and impaired phosphatidylserine exposure. Collagen and thrombin did not induce the release of cytochrome c nor the activation of caspase-3 and ROCK1. In contrast, in PLTs stored for more than 5 days, the phosphatidylserine exposure was associated with cytochrome c-induced caspase-3 and ROCK1 activation. ABT737, a BH3 mimetic that induces mitochondrial pathway of apoptosis, induced cytochrome c release and activation of caspase-3 and ROCK1 and phosphatidylserine exposure independent of CypD. CONCLUSION: These results show that in stored PLTs cytochrome c release and the subsequent activation of caspase-3 and ROCK1 mediate phosphatidylserine exposure and it is distinct from activation-induced phosphatidylserine exposure.

Lisinopril-Induced Angioedema in a Patient with Plasma Prekallikrein Deficiency.

Angiotensin-converting enzyme (ACE) inhibitors are extensively prescribed to treat patients with hypertension, congestive heart failure, and diabetic nephropathy. A small fraction of these patients (approximately 0.7%) develop angioedema, manifested by swelling of the lips and oropharynx. Angioedema of oropharynx is a medical emergency that can lead to asphyxiation and death. The angioedema is due to bradykinin generated from high molecular weight kininogen by kallikrein, which is derived from plasma prekallikrein by action of the factor XIIa, factor Xia, or prolylcarboxypeptidase. Bradykinin induces vasodilation and increased vascular permeability. ACE is the major degrading enzyme of bradykinin in the intravascular department. ACE inhibitors inhibit the proteolytic inactivation of bradykinin. We report a patient with oropharyngeal angioedema associated with an ACE inhibitor with complete absence of plasma prekallikrein due to homozygous mutation (Ser97PhefsTer173).