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Blood Advances Nov 2023Red blood cells (RBCs) and platelets contribute to the coagulation capacity in bleeding and thrombotic disorders. The thrombin generation (TG) process is considered to...
Red blood cells (RBCs) and platelets contribute to the coagulation capacity in bleeding and thrombotic disorders. The thrombin generation (TG) process is considered to reflect the interactions between plasma coagulation and the various blood cells. Using a new high-throughput method capturing the complete TG curve, we were able to compare TG in whole blood and autologous platelet-rich and platelet-poor plasma to redefine the blood cell contributions to the clotting process. We report a faster and initially higher generation of thrombin and shorter coagulation time in whole blood than in platelet-rich plasma upon low concentrations of coagulant triggers, including tissue factor, Russell viper venom factor X, factor Xa, factor XIa, and thrombin. The TG was accelerated with increased hematocrit and delayed after prior treatment of RBC with phosphatidylserine-blocking annexin A5. RBC treatment with ionomycin increased phosphatidylserine exposure, confirmed by flow cytometry, and increased the TG process. In reconstituted blood samples, the prior selective blockage of phosphatidylserine on RBC with annexin A5 enhanced glycoprotein VI-induced platelet procoagulant activity. For patients with anemia or erythrocytosis, cluster analysis revealed high or low whole-blood TG profiles in specific cases of anemia. The TG profiles lowered upon annexin A5 addition in the presence of RBCs and thus were determined by the extent of phosphatidylserine exposure of blood cells. Profiles for patients with polycythemia vera undergoing treatment were similar to that of control subjects. We concluded that RBC and platelets, in a phosphatidylserine-dependent way, contribute to the TG process. Determination of the whole-blood hypo- or hyper-coagulant activity may help to characterize a bleeding or thrombosis risk.
Topics: Humans; Thrombin; Phosphatidylserines; Annexin A5; Erythrocytes; Thrombosis; Coagulants; Anemia
PubMed: 37648671
DOI: 10.1182/bloodadvances.2023010027 -
Nature Communications Oct 2023Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients....
Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients. Host CO concentrations are 100-fold higher than the external environment and strains unable to grow at host CO concentrations are not pathogenic. Using a genetic screening and transcriptional profiling approach, we report that the TOR pathway is critical for C. neoformans adaptation to host CO partly through Ypk1-dependent remodeling of phosphatidylserine asymmetry at the plasma membrane. We also describe a C. neoformans ABC/PDR transporter (PDR9) that is highly expressed in CO-sensitive environmental strains, suppresses CO-induced phosphatidylserine/phospholipid remodeling, and increases susceptibility to host concentrations of CO. Interestingly, regulation of plasma membrane lipid asymmetry by the TOR-Ypk1 axis is distinct in C. neoformans compared to S. cerevisiae. Finally, host CO concentrations suppress the C. neoformans pathways that respond to host temperature (Mpk1) and pH (Rim101), indicating that host adaptation requires a stringent balance among distinct stress responses.
Topics: Humans; Cryptococcus neoformans; Saccharomyces cerevisiae; Phospholipids; Carbon Dioxide; Phosphatidylserines; Cryptococcosis; ATP-Binding Cassette Transporters
PubMed: 37852972
DOI: 10.1038/s41467-023-42318-y -
Cellular and Molecular Life Sciences :... Oct 2023The hepatitis E virus (HEV) is an underestimated RNA virus of which the viral life cycle and pathogenicity remain partially understood and for which specific antivirals...
The hepatitis E virus (HEV) is an underestimated RNA virus of which the viral life cycle and pathogenicity remain partially understood and for which specific antivirals are lacking. The virus exists in two forms: nonenveloped HEV that is shed in feces and transmits between hosts; and membrane-associated, quasi-enveloped HEV that circulates in the blood. It is suggested that both forms employ different mechanisms for cellular entry and internalization but little is known about the exact mechanisms. Interestingly, the membrane of enveloped HEV is enriched with phosphatidylserine, a natural ligand for the T-cell immunoglobulin and mucin domain-containing protein 1 (TIM1) during apoptosis and involved in 'apoptotic mimicry', a process by which viruses hijack the apoptosis pathway to promote infection. We here investigated the role of TIM1 in the entry process of HEV. We determined that HEV infection with particles derived from culture supernatant, which are cloaked by host-derived membranes (eHEV), was significantly impaired after knockout of TIM1, whereas infection with intracellular HEV particles (iHEV) was unaffected. eHEV infection was restored upon TIM1 expression; and enhanced after ectopic TIM1 expression. The significance of TIM1 during entry was further confirmed by viral binding assay, and point mutations of the PS-binding pocket diminished eHEV infection. In addition, Annexin V, a PS-binding molecule also significantly reduced infection. Taken together, our findings support a role for TIM1 in eHEV-mediated cell entry, facilitated by the PS present on the viral membrane, a strategy HEV may use to promote viral spread throughout the infected body.
Topics: Hepatitis E virus; Virus Internalization; Receptors, Cell Surface; Viruses
PubMed: 37833515
DOI: 10.1007/s00018-023-04977-4 -
Nature Structural & Molecular Biology Nov 2023Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined...
Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined to rapidly nucleate vesicles at the site of membrane damage, but the underlying molecular mechanisms remain poorly understood. Here we present the structure of Mus musculus TRIM72, a complete model of a TRIM E3 ubiquitin ligase. We demonstrated that the interaction between TRIM72 and phosphatidylserine-enriched membranes is necessary for its oligomeric assembly and ubiquitination activity. Using cryogenic electron tomography and subtomogram averaging, we elucidated a higher-order model of TRIM72 assembly on the phospholipid bilayer. Combining structural and biochemical techniques, we developed a working molecular model of TRIM72, providing insights into the regulation of RING-type E3 ligases through the cooperation of multiple domains in higher-order assemblies. Our findings establish a fundamental basis for the study of TRIM E3 ligases and have therapeutic implications for diseases associated with membrane repair.
Topics: Mice; Humans; Animals; Ubiquitin-Protein Ligases; Ubiquitination; Tripartite Motif Proteins; Heart Diseases; Models, Molecular; Membrane Proteins
PubMed: 37770719
DOI: 10.1038/s41594-023-01111-7 -
Journal of Pharmaceutical Health Care... May 2024Coronary heart disease, also known as ischemic heart disease, is induced by atherosclerosis, which is initiated by subendothelial retention of lipoproteins. Plasma... (Review)
Review
Coronary heart disease, also known as ischemic heart disease, is induced by atherosclerosis, which is initiated by subendothelial retention of lipoproteins. Plasma lipoproteins, including high density lipoprotein, low density lipoprotein (LDL), very low density lipoprotein, and chylomicron, are composed of a surface monolayer containing phospholipids and cholesterol and a hydrophobic core containing triglycerides and cholesteryl esters. Phospholipids play a crucial role in the binding of apolipoproteins and enzymes to lipoprotein surfaces, thereby regulating lipoprotein metabolism. High LDL-cholesterol is a well-known risk factor for coronary heart disease, and statins reduce the risk of coronary heart disease by lowering LDL-cholesterol levels. In contrast, the relationships of phospholipids in plasma lipoproteins with coronary heart disease have not yet been established. To further clarify the physiological and pathological roles of phospholipids, we have developed the simple high-throughput assays for quantifying all major phospholipid classes, namely phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol + cardiolipin, and sphingomyelin, using combinations of specific enzymes and a fluorogenic probe. These enzymatic fluorometric assays will be helpful in elucidating the associations between phospholipid classes in plasma lipoproteins and coronary heart disease and in identifying phospholipid biomarkers. This review describes recent progress in the identification of phospholipid biomarkers of coronary heart disease.
PubMed: 38734675
DOI: 10.1186/s40780-024-00344-y -
Cytometry. Part a : the Journal of the... Dec 2023Using spectral flow cytometry, we developed a 16-color panel for analysis of platelet phenotype and function in human whole blood. The panel contains markers of clinical...
Using spectral flow cytometry, we developed a 16-color panel for analysis of platelet phenotype and function in human whole blood. The panel contains markers of clinical relevance and follows an optimized protocol for the high-parameter phenotyping of (phosphatidylserine positive) procoagulant platelets. Inclusion of established markers, such as CD62P and PAC-1, allows the subsetting of classic (proinflammatory and proaggregatory) phenotypes, while addition of novel markers, such as TLR9, allows the resolution of platelets with nonclassic functions. Multiple inducible (C3b, CD63, CD107a, CD154, and TLT-1) and constitutive (CD29, CD31, CD32, CD36, CD42a, CD61, and GPVI) markers are also measurable, and we demonstrate the use of automatic gating for platelet analysis. The panel is widely applicable to research and clinical settings and can be readily modified, should users wish to tailor the panel to more specific needs.
Topics: Humans; Flow Cytometry; Blood Platelets; CD40 Ligand; Platelet Activation
PubMed: 37786346
DOI: 10.1002/cyto.a.24797 -
Rheumatology (Oxford, England) Feb 2024In 2006, at a meeting in Sydney, Australia, consensus was reached by an international group of specialists to establish a number of serological criteria that identify... (Review)
Review
In 2006, at a meeting in Sydney, Australia, consensus was reached by an international group of specialists to establish a number of serological criteria that identify patients with a history of thrombosis or pregnancy complications as having antiphospholipid syndrome (APS). These criteria were originally formulated for research purposes and to compare clinical trials in different centres. However, these same criteria are now generally used and accepted for the diagnosis and treatment of patients. The practice of using these criteria for direct patient care requires that these criteria are based on sound scientific evidence. Indeed, for all the autoantibodies that are officially included in the serological criteria, it has been shown that they induce thrombosis and fetal loss when infused into mice. There are also a number of additional autoantibodies that have been identified in these patients but for these antibodies there was not enough evidence to meet the official APS criteria in 2006. Seventeen years have now passed since the consensus meeting, therefore, this review examines whether additional studies performed with these 'non-criteria' autoantibodies have provided sufficient results to suggest the inclusion of these autoantibodies in the official serological criteria of APS.
Topics: Pregnancy; Female; Humans; Animals; Mice; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Autoantibodies; Thrombosis; Prenatal Care; Prothrombin
PubMed: 38320588
DOI: 10.1093/rheumatology/kead632 -
Blood Jan 2024Cell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase...
Cell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.
Topics: Female; Humans; Anemia, Hemolytic, Congenital; Calcium; Erythrocytes; Hydrops Fetalis; Ion Channels; Phospholipid Transfer Proteins
PubMed: 38033286
DOI: 10.1182/blood.2023021465 -
Immunological Reviews Oct 2023Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal... (Review)
Review
Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal pigment epithelial (RPE). It is a strictly rhythmic, synchronized process that underlies in part circadian regulation. We highlight findings from recently developed methods that quantify distinct phases of outer segment renewal in retinal tissue. At light onset, outer segments expose the conserved "eat-me" signal phosphatidylserine exclusively at their distal, most aged tip. A coordinated two-receptor efferocytosis process follows, in which ligands bridge outer segment phosphatidylserine with the RPE receptors αvβ5 integrin, inducing cytosolic signaling toward Rac1 and focal adhesion kinase/MERTK, and with MERTK directly, additionally inhibiting RhoA/ROCK and thus enabling F-actin dynamics favoring outer segment fragment engulfment. Photoreceptors and RPE persist for life with each RPE cell in the eye servicing dozens of overlying photoreceptors. Thus, RPE cells phagocytose more often and process more material than any other cell type. Mutant mice with impaired outer segment renewal largely retain functional photoreceptors and retinal integrity. However, when anti-inflammatory signaling in the RPE via MERTK or the related TYRO3 is lacking, catastrophic inflammation leads to immune cell infiltration that swiftly destroys the retina causing blindness.
Topics: Mice; Animals; Humans; c-Mer Tyrosine Kinase; Receptor Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Retinal Pigments; Phosphatidylserines; Retina; Phagocytosis; Inflammation; Mammals
PubMed: 37555340
DOI: 10.1111/imr.13264 -
Cell Death & Disease Jul 2023Apoptosis of endothelial cells prompts the release of apoptotic exosome-like vesicles (ApoExos), subtype extracellular vesicles secreted by apoptotic cells after...
Apoptosis of endothelial cells prompts the release of apoptotic exosome-like vesicles (ApoExos), subtype extracellular vesicles secreted by apoptotic cells after caspase-3 activation. ApoExos are different from both apoptotic bodies and classical exosomes in their protein and nucleic acid contents and functions. In contrast to classical apoptotic bodies, ApoExos induce immunogenic responses that can be maladaptive when not tightly regulated. In the present study, we elucidated the mechanisms by which ApoExos are internalized by endothelial cells, which leads to shared specific and functional mRNAs of importance to endothelial function. Using flow cytometry and confocal microscopy, we revealed that ApoExos were actively internalized by endothelial cells. SiRNA-induced inhibition of classical endocytosis pathways with pharmacological inhibitors showed that ApoExos were internalized via phosphatidylserine-dependent macropinocytosis independently of classical endocytosis pathways. An electron microscopy analysis revealed that ApoExos increased the macropinocytosis rate in endothelial cells, setting in motion a positive feedback loop that increased the amount of internalized ApoExos. Deep sequencing of total RNA revealed that ApoExos possessed a unique protein-coding RNA profile, with PCSK5 being the most abundant mRNA. Internalization of ApoExos by cells led to the transfer of this RNA content from the ApoExos to cells. Specifically, PCSK5 mRNA was transferred to cells that had taken up ApoExos, and these cells subsequently expressed PCSK5. Collectively, our findings suggest that macropinocytosis is an effective entry pathway for the delivery of RNAs carried by ApoExos and that these RNAs are functionally expressed by the endothelial cells that internalize them. As ApoExos express a specific mRNA signature, these results suggest new avenues to understand how ApoExos produced at sites of vascular injury impact vascular function.
Topics: Exosomes; Endothelial Cells; RNA, Messenger; Phosphatidylserines; RNA, Small Interfering
PubMed: 37474514
DOI: 10.1038/s41419-023-05991-x