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.

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.

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.

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.

Thrombotic, inflammatory, and HIF-regulated genes and thrombosis risk in polycythemia vera and essential thrombocythemia.

Thrombosis is a major cause of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET). The pathophysiology of thrombosis in these disorders remains unclear, and we hypothesized that upregulation of thrombotic, inflammatory, and hypoxia-inducible factor (HIF)-regulated genes may play a role in it. We performed unbiased RNA sequencing in granulocytes and platelets of PV patients and found differential expression of several thrombotic, inflammatory, and HIF-regulated genes. The expression of many of these genes positively correlated with JAK2 expression and JAK2V617F allelic burden. We then validated these findings by quantitative polymerase chain reaction analyses of selected gene transcripts in a larger number of PV and ET granulocytes and platelets (58 patients) and in 28 controls, and we compared these findings in patients with and without thrombosis. The study included 29 females and 29 males; of these, 28 had a history of thrombosis. We found that transcripts of several selected genes were upregulated in patients with PV or ET compared with controls. In granulocytes, the expression levels of F3, SELP, VEGFA, and SLC2A1 were significantly higher in patients with a history of thrombosis compared with those who did not have thrombosis. Patients with a history of thrombosis have significantly higher expression of IL1RAP (P < .05) in platelets compared with those without thrombosis. Our study confirms the presence of a thrombo-inflammatory state and augmented HIF activity in PV and ET and its role in thrombosis. These data may provide the background for targeted therapies in PV and ET.

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.

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).

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.

Ticagrelor induces paraoxonase-1 (PON1) and better protects hypercholesterolemic mice against atherosclerosis compared to clopidogrel.

Ticagrelor (TIC), a P2Y purinoceptor 12 (P2Y12)-receptor antagonist, has been widely used to treat patients with acute coronary syndrome. Although animal studies suggest that TIC protects against atherosclerosis, it remains unknown whether it does so through its potent platelet inhibition or through other pathways. Here, we placed hypercholesterolemic Ldlr-/-Apobec1-/- mice on a high-fat diet and treated them with either 25 mg/kg/day of clopidogrel (CLO) or 180 mg/kg/day of TIC for 16 weeks and evaluated the extent of atherosclerosis. Both treatments equally inhibited platelets as determined by ex vivo platelet aggregation assays. The extent of atherosclerosis, however, was significantly less in the TIC group than in the CLO group. Immunohistochemical staining and ELISA showed that TIC treatment was associated with less macrophage infiltration to the atherosclerotic intima and lower serum levels of CCL4, CXCL10, and TNFα, respectively, than CLO treatment. Treatment with TIC, but not CLO, was associated with higher serum activity and tissue level of paraoxonase-1 (PON1), an anti-atherosclerotic molecule, suggesting that TIC might exert greater anti-atherosclerotic activity, compared with CLO, through its unique ability to induce PON1. Although further studies are needed, TIC may prove to be a viable strategy in the prevention and treatment of chronic stable human atherosclerosis.

Wdr-1 is essential for F-actin interaction with focal adhesions in platelets.

: Wdr-1, an actin interacting protein, enhances cofilin's capacity to accelerate depolymerization of F-actin filaments. Wdr-1-deficient mice have impaired hemostasis due to defective inside-out integrin signaling in platelets. Here, we studied the role of Wdr-1 on outside-in signaling necessary for retraction of the clot and platelet spreading. Outside-in signaling was assessed by fibrin clot retraction assay and by adhesion and spreading of unstimulated platelets on fibrinogen substrate. The spatial distribution of actin, cofilin-1 and Wdr-1 were determined by immunofluorescence microscopy. Interaction of F-actin with focal adhesion kinase was assessed in dual-color confocal images and by immunoblotting of F-actin filaments. Clot retraction is markedly impaired in Wdr-1-deficient platelets. Wdr-1-deficient platelets adhere and spread poorly on fibrinogen substrate compared with wild-type controls. In resting platelets, Wdr-1 is colocalized with cofilin-1 in cortical actin. Following platelets spreading on fibrinogen substrate, Wdr-1 translocates to the cytoskeleton in association with cofilin-1. In Wdr-1-deficient platelets, cofilin-1 is aberrantly localized throughout the cytoplasm and there is no significant change following adhesion to fibrinogen substrate. The actin filaments formed upon spreading on fibrinogen are mostly in the periphery of the platelets and does not traverse the cytoplasm. Furthermore, there is diminished colocalization of actin filaments with focal adhesion kinase. These studies show that Wdr-1 is essential for the localization of cofilin-1 to the platelet membrane skeleton. F-actin fails to attach to focal adhesions resulting in defective reorganization of actin filaments necessary for platelet spreading and clot retraction.

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.

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.

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.

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.

Wdr1-Dependent Actin Reorganization in Platelet Activation.

In resting platelets, the integrin αIIbß3 is present in a low-affinity "bent" state. During platelet aggregation, intracytoplasmic signals induce conformational changes (inside-out signaling) that result in a "swung-out" conformation competent to bind ligands such as fibrinogen. The cytoskeleton plays an essential role in αIIbß3 activation. We investigated the role of the actin interacting protein Wdr1 in αIIbß3 activation. Wdr1-hypomorphic mice had a prolonged bleeding time (> 10 minutes) compared to that of wild-type mice (2.1 ± 0.7 minutes). Their platelets had impaired aggregation to collagen and thrombin. In a FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1-hypomorphic mice was prolonged significantly compared to wild-type mice (9.0 ± 10.5 minutes versus 5.8 ± 12.6 minutes (p = 0.041). Activation-induced binding of JON/A (a conformation-specific antibody to activated αIIbß3) was significantly less in Wdr1-hypomorphic platelets at various concentrations of collagen, indicating impaired inside-out activation of αIIbß3, despite a normal calcium response. Actin turnover, assessed by measuring F-actin and G-actin ratios during collagen- and thrombin-induced platelet aggregation, was highly impaired in Wdr1-hypomorphic platelets. Furthermore, talin failed to redistribute and translocate to the cytoskeleton following activation in Wdr1-hypomorphic platelets. These studies show that Wdr1 is essential for talin-induced activation of αIIbß3 during platelet activation.

Lipid profile of platelets and platelet-derived microparticles in ovarian cancer.

BACKGROUND: Ovarian cancer patients have a high risk of developing venous thrombosis. The membrane lipid bilayer of platelets and platelet-derived microparticles (PMP) provides a platform for assembly of coagulation proteins and generation of blood clots. METHODS: We compared the lipid composition of platelets and PMPs in patients with ovarian cancer to those in healthy subjects. We used shotgun lipidomics to quantify 12 classes and 177 species of lipids. RESULTS: We found a significant change in 2 classes of lipids in platelets and PMPs isolated from ovarian cancer patients: higher phosphatidylinositol and lower lyso-phosphatidylcholine. The level of 28 species of lipids was also significantly altered in the direction of an increase in the pro-coagulant and a reduction in the anticoagulant lipids. We found that cancer platelets expressed less lipid phosphate phosphatase 1 (LPP1), a key enzyme in phospholipid biosynthesis pathways, than normal platelets. The reduction in LPP1 might contribute to the changes in the lipid profile of cancer platelets. CONCLUSION: Our results support a procoagulant lipid profile of platelets in ovarian cancer patients that can play a role in the increased risk of venous thrombosis in these patients. GENERAL SIGNIFICANCE: As far as we are aware, our study is the first study on platelet lipidomics in ovarian cancer. The importance of our findings for the future studies are: 1) a similar change in lipid profile of platelets and PMP may be responsible for hypercoagulability in other cancers, and 2) plasma level of high-risk lipids for venous thrombosis may be useful biomarkers.

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.