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MedRxiv : the Preprint Server For... Oct 2023Coagulopathy and associated bleeding and venous thromboembolism (VTE) are major causes of morbidity and mortality in patients with acute leukemia. The underlying...
BACKGROUND
Coagulopathy and associated bleeding and venous thromboembolism (VTE) are major causes of morbidity and mortality in patients with acute leukemia. The underlying mechanisms of these complications have not been fully elucidated.
OBJECTIVES
To evaluate the associations between biomarker levels and bleeding and VTE in acute leukemia patients.
PATIENTS/METHOD
We examined plasma levels of activators, inhibitors and biomarkers of the coagulation and fibrinolytic pathways in patients ≥18 years with newly diagnosed acute leukemia compared to healthy controls. Multivariable regression models were used to examine the association of biomarkers with bleeding and VTE in acute leukemia patients. The study included 358 patients with acute leukemia (29 acute promyelocytic leukemia [APL], 253 non-APL acute myeloid leukemia [AML] and 76 acute lymphoblastic leukemia [ALL]), and 30 healthy controls.
RESULTS
Patients with acute leukemia had higher levels of extracellular vesicle (EV) tissue factor (TF) activity, phosphatidylserine-positive EVs, plasminogen activator inhibitor-1 (PAI-1), plasmin-antiplasmin complexes, cell-free DNA and lower levels of citrullinated histone H3-DNA complexes compared to healthy controls. APL patients had the highest levels of EVTF activity and the lowest levels of tissue plasminogen activator among the acute leukemia patients. There were 41 bleeding and 37 VTE events in acute leukemia patients. High EVTF activity was associated with increased risk of bleeding (sHR 2.30, 95%CI 0.99-5.31) whereas high PAI-1 was associated with increased risk of VTE (sHR 3.79, 95%CI 1.40-10.28) in these patients.
CONCLUSIONS
Our study shows alterations in several biomarkers in acute leukemia and identifies biomarkers associated with risk of bleeding and VTE.
PubMed: 37905148
DOI: 10.1101/2023.10.18.23297216 -
Thrombosis and Haemostasis Dec 2023Although thrombosis events are the leading complication of uremia, their mechanism is largely unknown. The interaction between endothelial cells (ECs) and red blood...
BACKGROUND
Although thrombosis events are the leading complication of uremia, their mechanism is largely unknown. The interaction between endothelial cells (ECs) and red blood cells (RBCs) in uremic solutes and its prothrombotic role need to be investigated.
METHODS AND RESULTS
Here, we established an in vitro co-incubation model of uremic RBC and EC as well as a uremic rat model induced by adenine. Using flow cytometry, confocal microscopy, and electron microscopy, we found increased erythrophagocytosis by EC accompanied by increased reactive oxygen species, lipid peroxidation, and impairment of mitochondria, indicating that ECs undergo ferroptosis. Further investigations showed increased proteins' expression of heme oxygenase-1 and ferritin and labile iron pool accumulation in EC, which could be suppressed by deferoxamine (DFO). The ferroptosis-negative regulators glutathione peroxidase 4 and SLC7A11 were decreased in our erythrophagocytosis model and could be enhanced by ferrostatin-1 or DFO. In vivo, we observed that vascular EC phagocytosed RBC and underwent ferroptosis in the kidney of the uremic rat, which could be inhibited by blocking the phagocytic pathway or inhibiting ferroptosis. Next, we found that the high tendency of thrombus formation was accompanied by erythrophagocytosis-induced ferroptosis in vitro and in vivo. Importantly, we further revealed that upregulated TMEM16F expression mediated phosphatidylserine externalization on ferroptotic EC, which contributed to a uremia-associated hypercoagulable state.
CONCLUSION
Our results indicate that erythrophagocytosis-triggered ferroptosis followed by phosphatidylserine exposure of EC may play a key role in uremic thrombotic complications, which may be a promising target to prevent thrombogenesis of uremia.
Topics: Rats; Animals; Ferroptosis; Endothelial Cells; Phosphatidylserines; Erythrocytes; Thrombosis; Uremia
PubMed: 37364609
DOI: 10.1055/a-2117-7890 -
Journal of Thrombosis and Haemostasis :... Dec 2023Our recent studies showed that activated factor (F) VII (FVIIa) releases extracellular vesicles (EVs) from the endothelium. FVIIa-released EVs were found to be enriched...
BACKGROUND
Our recent studies showed that activated factor (F) VII (FVIIa) releases extracellular vesicles (EVs) from the endothelium. FVIIa-released EVs were found to be enriched with phosphatidylserine (PS) and contribute to the hemostatic effect of FVIIa in thrombocytopenia and hemophilia.
OBJECTIVE
To investigate mechanisms by which FVIIa induces EV biogenesis and enriches EVs with PS.
METHODS
FVIIa activation of acid sphingomyelinase (aSMase) was evaluated by its translocation to the cell surface. The role of aSMase in the biogenesis of FVIIa-induced EVs and their enrichment with PS was investigated using specific siRNAs and inhibitors of aSMase and its downstream metabolites. Wild-type and aSMase mice were injected with a control vehicle or FVIIa. EVs released into circulation were quantified by nanoparticle tracking analysis. EVs hemostatic potential was assessed in a murine thrombocytopenia model.
RESULTS
FVIIa activation of aSMase is responsible for both the externalization of PS and the release of EVs in endothelial cells. FVIIa-induced aSMase activation led to ceramide generation and de novo expression of transmembrane protein 16F. Inhibitors of ceramidases, sphingosine kinase, or sphingosine-1-phosphate receptor modulator blocked FVIIa-induced expression of transmembrane protein 16F and PS externalization without interfering with FVIIa release of EVs. In vivo, FVIIa release of EVs was markedly impaired in aSMase mice compared with wild-type mice. Administration of a low dose of FVIIa, sufficient to induce EVs release, corrected bleeding associated with thrombocytopenia in wild-type mice but not in aSMase mice.
CONCLUSION
Our study identifies a novel mechanism by which FVIIa induces PS externalization and releases PS-enriched EVs.
Topics: Animals; Mice; Endothelial Cells; Extracellular Vesicles; Factor VIIa; Hemostatics; Phosphatidylserines; Sphingomyelin Phosphodiesterase; Thrombocytopenia
PubMed: 37875382
DOI: 10.1016/j.jtha.2023.08.025 -
Nature Microbiology Apr 2024Some viruses are rarely transmitted orally or sexually despite their presence in saliva, breast milk, or semen. We previously identified that extracellular vesicles...
Some viruses are rarely transmitted orally or sexually despite their presence in saliva, breast milk, or semen. We previously identified that extracellular vesicles (EVs) in semen and saliva inhibit Zika virus infection. However, the antiviral spectrum and underlying mechanism remained unclear. Here we applied lipidomics and flow cytometry to show that these EVs expose phosphatidylserine (PS). By blocking PS receptors, targeted by Zika virus in the process of apoptotic mimicry, they interfere with viral attachment and entry. Consequently, physiological concentrations of EVs applied in vitro efficiently inhibited infection by apoptotic mimicry dengue, West Nile, Chikungunya, Ebola and vesicular stomatitis viruses, but not severe acute respiratory syndrome coronavirus 2, human immunodeficiency virus 1, hepatitis C virus and herpesviruses that use other entry receptors. Our results identify the role of PS-rich EVs in body fluids in innate defence against infection via viral apoptotic mimicries, explaining why these viruses are primarily transmitted via PS-EV-deficient blood or blood-ingesting arthropods rather than direct human-to-human contact.
Topics: Female; Humans; Phosphatidylserines; Viruses; Extracellular Vesicles; Virus Attachment; Zika Virus; Body Fluids; Zika Virus Infection
PubMed: 38528146
DOI: 10.1038/s41564-024-01637-6 -
Nature Communications Jan 2024Efferocytic clearance of apoptotic cells in general, and T cells in particular, is required for tissue and immune homeostasis. Transmembrane mucins are extended...
Efferocytic clearance of apoptotic cells in general, and T cells in particular, is required for tissue and immune homeostasis. Transmembrane mucins are extended glycoproteins highly expressed in the cell glycocalyx that function as a barrier to phagocytosis. Whether and how mucins may be regulated during cell death to facilitate efferocytic corpse clearance is not well understood. Here we show that normal and transformed human T cells express a subset of mucins which are rapidly and selectively removed from the cell surface during apoptosis. This process is mediated by the ADAM10 sheddase, the activity of which is associated with XKR8-catalyzed flipping of phosphatidylserine to the outer leaflet of the plasma membrane. Mucin clearance enhances uptake of apoptotic T cells by macrophages, confirming mucins as an enzymatically-modulatable barrier to efferocytosis. Together these findings demonstrate a glycocalyx regulatory pathway with implications for therapeutic intervention in the clearance of normal and transformed apoptotic T cells.
Topics: Humans; Efferocytosis; Mucins; T-Lymphocytes; Apoptosis; Phagocytosis; ADAM10 Protein; Membrane Proteins; Amyloid Precursor Protein Secretases
PubMed: 38225245
DOI: 10.1038/s41467-023-44619-8 -
Basic Research in Cardiology Aug 2023While low concentrations of high-density lipoprotein-cholesterol (HDL-C) are widely accepted as an independent cardiovascular risk factor, HDL-C-rising therapies largely...
While low concentrations of high-density lipoprotein-cholesterol (HDL-C) are widely accepted as an independent cardiovascular risk factor, HDL-C-rising therapies largely failed, suggesting the importance of both HDL functions and individual subspecies. Indeed HDL particles are highly heterogeneous, with small, dense pre-beta-HDLs being considered highly biologically active but remaining poorly studied, largely reflecting difficulties for their purification. We developed an original experimental approach allowing the isolation of sufficient amounts of human pre-beta-HDLs and revealing the specificity of their proteomic and lipidomic profiles and biological activities. Pre-beta-HDLs were enriched in highly poly-unsaturated species of phosphatidic acid and phosphatidylserine, and in an unexpectedly high number of proteins implicated in the inflammatory response, including serum paraoxonase/arylesterase-1, vitronectin and clusterin, as well as in complement regulation and immunity, including haptoglobin-related protein, complement proteins and those of the immunoglobulin class. Interestingly, amongst proteins associated with lipid metabolism, phospholipid transfer protein, cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase were strongly enriched in, or restricted to, pre-beta-HDL. Furthermore, pre-beta-HDL potently mediated cellular cholesterol efflux and displayed strong anti-inflammatory activities. A correlational network analysis between lipidome, proteome and biological activities highlighted 15 individual lipid and protein components of pre-beta-HDL relevant to cardiovascular disease, which may constitute novel diagnostic targets in a pathological context of altered lipoprotein metabolism.
Topics: Humans; Cardiovascular Diseases; Proteomics; Cholesterol, HDL; Heart Disease Risk Factors; Lipid Metabolism
PubMed: 37639039
DOI: 10.1007/s00395-023-01004-2 -
Life (Basel, Switzerland) Dec 2023Eryptosis stimulated by anticancer drugs can lead to anemia in patients. β-caryophyllene oxide (CPO) is an anticancer sesquiterpene present in various plants; however,...
BACKGROUND
Eryptosis stimulated by anticancer drugs can lead to anemia in patients. β-caryophyllene oxide (CPO) is an anticancer sesquiterpene present in various plants; however, its effect on the structure and function of human red blood cells (RBCs) remains unexplored. The aim of this study was to investigate the hemolytic and eryptotic activities and underlying molecular mechanisms of CPO in human RBCs.
METHODS
Cells were treated with 10-100 μM of CPO for 24 h at 37 °C, and hemolysis, LDH, AST, and AChE activities were photometrically assayed. Flow cytometry was employed to determine changes in cell volume from FSC, phosphatidylserine (PS) externalization by annexin-V-FITC, intracellular calcium by Fluo4/AM, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (HDCFDA). Cells were also cotreated with CPO and specific signaling inhibitors and antihemolytic agents. Furthermore, whole blood was exposed to CPO to assess its toxicity to other peripheral blood cells.
RESULTS
CPO induced concentration-responsive hemolysis with LDH and AST leakage, in addition to PS exposure, cell shrinkage, Ca accumulation, oxidative stress, and reduced AChE activity. The toxicity of CPO was ameliorated by D4476, staurosporin, and necrosulfonamide. ATP and PEG 8000 protected the cells from hemolysis, while urea and isotonic sucrose had opposite effects.
CONCLUSIONS
CPO stimulates hemolysis and eryptosis through energy depletion, Ca buildup, oxidative stress, and the signaling mediators casein kinase 1α, protein kinase C, and mixed lineage kinase domain-like pseudokinase. Development of CPO as an anticancer therapeutic must be approached with prudence to mitigate adverse effects on RBCs using eryptosis inhibitors, Ca channel blockers, and antioxidants.
PubMed: 38137900
DOI: 10.3390/life13122299 -
Journal of Thrombosis and Haemostasis :... Sep 2023The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against...
BACKGROUND
The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against β2-glycoprotein I (β2GPI) and prothrombin. LA is associated with activated protein C (APC) resistance, which might contribute to thrombotic risk in patients with antiphospholipid syndrome. How antibodies against β2GPI and prothrombin cause APC resistance is currently unclear.
OBJECTIVES
To investigate how anti-β2GPI and antiphosphatidylserine/prothrombin (PS/PT) antibodies induce APC resistance.
METHODS
The effects of anti-β2GPI and anti-PS/PT antibodies on APC resistance were studied in plasma (of patients with antiphospholipid syndrome) and with purified coagulation factors and antibodies.
RESULTS
APC resistance was observed in LA-positive patients with anti-β2GPI or anti-PS/PT antibodies and in normal plasma spiked with monoclonal anti-β2GPI or anti-PS/PT antibodies with LA activity. Analysis of factor (F)V cleavage patterns after APC incubation indicated that anti-β2GPI antibodies attenuated APC-mediated FV cleavage at R506 and R306. APC-mediated cleavage at R506 is required for FV cofactor activity during inactivation of FVIIIa. Assays with purified coagulation factors confirmed that anti-β2GPI antibodies interfered with the cofactor function of FV during FVIIIa inactivation but not with FVa inactivation. Anti-PS/PT antibodies attenuated APC-mediated FVa and FVIIIa inactivation. Analysis of FV(a) cleavage patterns after APC incubation indicated that anti-PS/PT antibodies interfere with APC-mediated cleavage of FV at positions R506 and R306.
CONCLUSION
Anti-β2GPI antibodies with LA activity contribute to a procoagulant state by causing APC resistance via interference with the cofactor function of FV during FVIIIa inactivation. LA-causing anti-PS/PT antibodies interfere with the anticoagulant function of APC by preventing FV(a) cleavage.
Topics: Humans; Activated Protein C Resistance; Antiphospholipid Syndrome; Autoantibodies; beta 2-Glycoprotein I; Factor V; Lupus Coagulation Inhibitor; Phosphatidylserines; Protein C; Prothrombin; Thrombosis
PubMed: 37290588
DOI: 10.1016/j.jtha.2023.05.024 -
Nature Communications Sep 2023The "eat me" signal, phosphatidylserine is exposed on the surface of dying cells by phospholipid scrambling. Previously, we showed that the Xkr family protein Xkr4 is...
The "eat me" signal, phosphatidylserine is exposed on the surface of dying cells by phospholipid scrambling. Previously, we showed that the Xkr family protein Xkr4 is activated by caspase-mediated cleavage and binding of the XRCC4 fragment. Here, we show that extracellular calcium is an additional factor needed to activate Xkr4. The constitutively active mutant of Xkr4 is found to induce phospholipid scrambling in an extracellular, but not intracellular, calcium-dependent manner. Importantly, other Xkr family members also require extracellular calcium for activation. Alanine scanning shows that D123 and D127 of TM1 and E310 of TM3 coordinate calcium binding. Moreover, lysine scanning demonstrates that the E310K mutation-mediated salt bridge between TM1 and TM3 bypasses the requirement of calcium. Cysteine scanning proves that disulfide bond formation between TM1 and TM3 also activates phospholipid scrambling without calcium. Collectively, this study shows that extracellular calcium functions as a molecular glue for TM1 and TM3 of Xkr proteins for activation, thus demonstrating a regulatory mechanism for multi-transmembrane region-containing proteins.
Topics: Calcium; Alanine; Biological Transport; Caspases; Phosphatidylserines
PubMed: 37696806
DOI: 10.1038/s41467-023-40934-2 -
Journal of Thrombosis and Haemostasis :... Aug 2023Procoagulant platelets are a subpopulation of highly activated platelets that promote coagulation through surface-exposed, negatively charged phospholipids, especially...
BACKGROUND
Procoagulant platelets are a subpopulation of highly activated platelets that promote coagulation through surface-exposed, negatively charged phospholipids, especially phosphatidylserine. Procoagulant platelets are important for clot stabilization during hemostasis, and an increased number of these platelets is associated with thrombotic risk. There is a need for harmonization in this area since many of the markers and methods used to assess procoagulant platelets are not specific when used in isolation but are also associated with platelet apoptosis.
OBJECTIVES
We initiated this project to identify a minimum set of markers and/or methods that can detect and distinguish procoagulant platelets from apoptotic platelets.
METHODS
The study design involved a primary panel with 27 international experts who participated in an online survey and moderated virtual focus group meetings. Primary and secondary panel members were then invited to provide input on themes and statements generated from the focus groups.
RESULTS
This led to a recommendation to use flow cytometry and a combination of the following 3 surface markers to differentiate procoagulant platelets from apoptotic platelets: P-selectin (CD62P), phosphatidylserine (recognized by annexin V), and the platelet-specific receptor GPIX (CD42a) or α integrin (CD41, GPIIb).
CONCLUSION
Procoagulant platelets are expected to be positive for all 3 markers, while apoptotic platelets are positive for annexin V and the platelet-specific surface receptor(s) but negative for P-selectin.
Topics: Humans; Blood Platelets; P-Selectin; Phosphatidylserines; Annexin A5; Consensus; Platelet Glycoprotein GPIb-IX Complex; Communication; Platelet Activation
PubMed: 37172731
DOI: 10.1016/j.jtha.2023.05.001