22 results on '"Gilbert GE"'
Search Results
2. Membrane curvature and PS localize coagulation proteins to filopodia and retraction fibers of endothelial cells.
- Author
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Carman CV, Nikova DN, Sakurai Y, Shi J, Novakovic VA, Rasmussen JT, Lam WA, and Gilbert GE
- Subjects
- Endothelial Cells metabolism, Factor Va chemistry, Factor Va metabolism, Pseudopodia metabolism, Fibrin, Thromboplastin metabolism, Phosphatidylserines metabolism
- Abstract
Prior reports indicate that the convex membrane curvature of phosphatidylserine (PS)-containing vesicles enhances formation of binding sites for factor Va and lactadherin. Yet, the relationship of convex curvature to localization of these proteins on cells remains unknown. We developed a membrane topology model, using phospholipid bilayers supported by nano-etched silica substrates, to further explore the relationship between curvature and localization of coagulation proteins. Ridge convexity corresponded to maximal curvature of physiologic membranes (radii of 10 or 30 nm) and the troughs had a variable concave curvature. The benchmark PS probe lactadherin exhibited strong differential binding to the ridges, on membranes with 4% to 15% PS. Factor Va, with a PS-binding motif homologous to lactadherin, also bound selectively to the ridges. Bound factor Va supported coincident binding of factor Xa, localizing prothrombinase complexes to the ridges. Endothelial cells responded to prothrombotic stressors and stimuli (staurosporine, tumor necrosis factor-α [TNF- α]) by retracting cell margins and forming filaments and filopodia. These had a high positive curvature similar to supported membrane ridges and selectively bound lactadherin. Likewise, the retraction filaments and filopodia bound factor Va and supported assembly of prothrombinase, whereas the cell body did not. The perfusion of plasma over TNF-α-stimulated endothelia in culture dishes and engineered 3-dimensional microvessels led to fibrin deposition at cell margins, inhibited by lactadherin, without clotting of bulk plasma. Our results indicate that stressed or stimulated endothelial cells support prothrombinase activity localized to convex topological features at cell margins. These findings may relate to perivascular fibrin deposition in sepsis and inflammation., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.) more...
- Published
- 2023
- Full Text
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3. Thrombotic Role of Blood and Endothelial Cells in Uremia through Phosphatidylserine Exposure and Microparticle Release.
- Author
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Gao C, Xie R, Yu C, Ma R, Dong W, Meng H, Zhang Y, Si Y, Zhang Z, Novakovic V, Zhang Y, Kou J, Bi Y, Li B, Xie R, Gilbert GE, Zhou J, and Shi J
- Subjects
- Adult, Aged, Animals, Blood Coagulation, Cattle, Coagulants, Factor Xa metabolism, Female, Fibrin metabolism, Flow Cytometry, Human Umbilical Vein Endothelial Cells, Humans, Male, Membrane Glycoproteins chemistry, Microscopy, Confocal, Middle Aged, Milk Proteins chemistry, Thrombin metabolism, Thromboplastin metabolism, Thrombosis prevention & control, Cell-Derived Microparticles metabolism, Endothelial Cells metabolism, Phosphatidylserines chemistry, Thrombosis blood, Thrombosis metabolism, Uremia blood
- Abstract
The mechanisms contributing to an increased risk of thrombosis in uremia are complex and require clarification. There is scant morphological evidence of membrane-dependent binding of factor Xa (FXa) and factor Va (FVa) on endothelial cells (EC) in vitro. Our objectives were to confirm that exposed phosphatidylserine (PS) on microparticle (MP), EC, and peripheral blood cell (PBC) has a prothrombotic role in uremic patients and to provide visible and morphological evidence of PS-dependent prothrombinase assembly in vitro. We found that uremic patients had more circulating MP (derived from PBC and EC) than controls. Additionally, patients had more exposed PS on their MPs and PBCs, especially in the hemodialysis group. In vitro, EC exposed more PS in uremic toxins or serum. Moreover, reconstitution experiments showed that at the early stages, PS exposure was partially reversible. Using confocal microscopy, we observed that PS-rich membranes of EC and MP provided binding sites for FVa and FXa. Further, exposure of PS in uremia resulted in increased generation of FXa, thrombin, and fibrin and significantly shortened coagulation time. Lactadherin, a protein that blocks PS, reduced 80% of procoagulant activity on PBC, EC, and MP. Our results suggest that PBC and EC in uremic milieu are easily injured or activated, which exposes PS and causes a release of MP, providing abundant procoagulant membrane surfaces and thus facilitating thrombus formation. Blocking PS binding sites could become a new therapeutic target for preventing thrombosis. more...
- Published
- 2015
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4. Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine.
- Author
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Gilbert GE, Novakovic VA, Shi J, Rasmussen J, and Pipe SW
- Subjects
- Binding Sites physiology, Cells, Cultured, Humans, Protein Binding physiology, Blood Platelets metabolism, Factor VIII metabolism, Fibrin metabolism, Phosphatidylserines metabolism, Platelet Activation physiology
- Abstract
Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the αIIbβ3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites. more...
- Published
- 2015
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5. Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome.
- Author
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Gao C, Xie R, Yu C, Wang Q, Shi F, Yao C, Xie R, Zhou J, Gilbert GE, and Shi J
- Subjects
- Adult, Antigens, Surface metabolism, Blood Platelets metabolism, Carrier Proteins, Case-Control Studies, Factor Xa biosynthesis, Female, Glomerulonephritis, Membranous metabolism, Humans, Intercellular Signaling Peptides and Proteins, Male, Microscopy, Confocal methods, Middle Aged, Milk Proteins metabolism, Nephrosis, Lipoid metabolism, Particle Size, Thromboplastin metabolism, Blood Platelets cytology, Coagulants metabolism, Erythrocytes metabolism, Glomerulonephritis, Membranous blood, Nephrosis, Lipoid blood, Phosphatidylserines metabolism
- Abstract
Recent studies showed that an imbalance of prothrombotic and antithrombotic factors and impaired thrombolytic activity contribute to the thrombophilia of the nephrotic syndrome (NS). However, it is not clear whether blood cell injury and/or activation is involved in hypercoagulability in NS patients. Our objectives were to study the increase in microparticle (MP) release and phosphatidylserine (PS) exposure on the outer membrane of MP-origin cells in NS patients, and to evaluate their procoagulant activity (PCA). The subjects were patients with membranous nephropathy (MN), minimal change nephrotic syndrome (MCNS) and healthy controls. Analyses of MPs and PS exposure were performed using a flow cytometer. PCA was determined by clotting time and purified coagulation complex assays. We found that lactadherin+ MPs, which derived from red blood cells (RBC), platelet and endothelial cell, increased in NS patients. Moreover, PS exposure on RBCs and platelets in each NS group, especially in MN, are higher than that in controls. MP shedding and PS exposure of RBCs/platelets were highly procoagulant in NS patients. However, blockade of PS with lactadherin inhibited over 90% of PCA while an anti-tissue factor antibody had no significant inhibition effect. Our results demonstrate that the thrombophilic susceptibility of NS may be partly ascribed to MP release and PS exposure of RBCs, platelets and endothelial cells. Lactadherin is a sensitive probe for PS that has high anticoagulant activity. more...
- Published
- 2012
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6. Lactadherin binds to phosphatidylserine-containing vesicles in a two-step mechanism sensitive to vesicle size and composition.
- Author
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Otzen DE, Blans K, Wang H, Gilbert GE, and Rasmussen JT
- Subjects
- Animals, Cattle, Kinetics, Membrane Glycoproteins chemistry, Micelles, Milk Proteins chemistry, Phosphatidylethanolamines metabolism, Protein Binding, Protein Structure, Tertiary, Spectrometry, Fluorescence, Membrane Glycoproteins metabolism, Milk Proteins metabolism, Phosphatidylserines metabolism, Unilamellar Liposomes chemistry
- Abstract
Lactadherin binds to phosphatidylserine (PS) in a stereospecific and calcium independent manner that is promoted by vesicle curvature. Because membrane binding of lactadherin is supported by a PS content of as little as 0.5%, lactadherin is a useful marker for cell stress where limited PS is exposed, as well as for apoptosis where PS freely traverses the plasma membrane. To gain further insight into the membrane-binding mechanism, we have utilized intrinsic lactadherin fluorescence. Our results indicate that intrinsic fluorescence increases and is blue-shifted upon membrane binding. Stopped-flow kinetic experiments confirm the specificity for PS and that the C2 domain contains a PS recognition motif. The stopped-flow kinetic data are consistent with a two-step binding mechanism, in which initial binding is followed by a slower step that involves either a conformational change or an altered degree of membrane insertion. Binding is detected at concentrations down to 0.03% PS and the capacity of binding reaches saturation around 1% PS (midpoint 0.15% PS). Higher concentrations of PS (and also to some extent PE) increase the association kinetics and the affinity. Increasing vesicle curvature promotes association. Remarkably, replacement of vesicles with micelles destroys the specificity for PS lipids. We conclude that the vesicular environment provides optimal conditions for presentation and recognition of PS by lactadherin in a simple binding mechanism. This article is part of a Special Issue entitled: Protein Folding in Membranes., (Copyright © 2011 Elsevier B.V. All rights reserved.) more...
- Published
- 2012
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7. Lactadherin functions as a probe for phosphatidylserine exposure and as an anticoagulant in the study of stored platelets.
- Author
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Hou J, Fu Y, Zhou J, Li W, Xie R, Cao F, Gilbert GE, and Shi J
- Subjects
- Annexin A5 chemistry, Antigens, Surface chemistry, Humans, Milk Proteins chemistry, Annexin A5 analysis, Anticoagulants, Antigens, Surface analysis, Milk Proteins analysis, Phosphatidylserines, Preservation, Biological
- Abstract
Background and Objectives: Annexin V, the long established standard method of measuring phosphatidylserine (PS) exposure, is not the most suitable probe for the study of stored platelets because of calcium dependence and low sensitivity under 8% PS exposure. The aim of this study was to show lactadherin as a sensitive probe for PS exposure and an effective anticoagulant for stored platelets., Materials and Methods: PS exposure and the associated procoagulant activity of platelets in 20 platelet concentrate units were investigated by flow cytometry, confocal microscopy, coagulation time analysis and enzymatic assays. Annexin V and lactadherin were utilized separately in this study., Results: Using lactadherin, we identified higher levels of PS exposure on the platelets and microparticles compared to detection using annexin V by flow cytometry. Lactadherin staining showed earlier PS exposure localized to platelets membrane in the earlier stage of storage. Subsequently, PS was distributed on the narrow rim of the plasma membrane and blebbing vesicles. Lactadherin or annexin V(32 nm) prolonged coagulation time 2·4 fold versus twofold. The production of thrombin and intrinsic/extrinsic factor Xase were inhibited approximately 85∼90% and 65∼70% by lactadherin and annexin V, respectively., Conclusion: Lactadherin can provide the quantification and location of PS exposure on banked platelets in the absence of agonists. Furthermore, lactadherin is a more effective anticoagulant for inhibiting the procoagulant activity of stored platelets compared with annexin V., (© 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.) more...
- Published
- 2011
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8. Daunorubicin induces procoagulant activity of cultured endothelial cells through phosphatidylserine exposure and microparticles release.
- Author
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Fu Y, Zhou J, Li H, Cao F, Su Y, Fan S, Li Y, Wang S, Li L, Gilbert GE, and Shi J
- Subjects
- Antibiotics, Antineoplastic toxicity, Anticoagulants pharmacology, Blood Coagulation Tests, Cell-Derived Microparticles metabolism, Cells, Cultured, Daunorubicin toxicity, Dose-Response Relationship, Drug, Endothelial Cells metabolism, Factor Xa metabolism, Flow Cytometry, Humans, Membrane Glycoproteins pharmacology, Milk Proteins pharmacology, Thrombin metabolism, Thromboplastin metabolism, Thrombosis blood, Thrombosis chemically induced, Antibiotics, Antineoplastic pharmacology, Blood Coagulation drug effects, Cell-Derived Microparticles drug effects, Daunorubicin pharmacology, Endothelial Cells drug effects, Phosphatidylserines metabolism
- Abstract
Administration of various chemotherapeutic agents is associated with an increased risk of thrombotic events. Although vascular endothelium plays a predominant role in blood coagulation and fibrinolysis, the effect of cytotoxic drugs on the procoagulant activity (PCA) of endothelial cells has not been well evaluated. Our study aims to investigate the possibility that daunorubicin, a chemotherapeutic agent, exerts prothrombotic effect on endothelial cells. We tested the impact of daunorubicin on phosphatidylserine (PS) exposure, endothelial microparticles (EMPs) release and consequent PCA. Human umbilical vein endothelial cells (HUVECs) were treated with daunorubicin (0.1, 0.2, 0.5, 1, 2 mM) for 24 hours. PCA of HUVECs was measured using clotting time and purified coagulation complex assays. Counts and PCA of EMPs were evaluated by flow cytometry and clotting time assay, respectively. Lactadherin was used as a novel probe for detection of PS exposure and EMPs release. We found that daunorubicin dose-dependently increased the PS exposure and consequent PCA of HUVECs. Moreover, daunorubicin treatment also enhanced the release of EMPs which were highly procoagulant. This increment was especially significant at 0.2 mM of daunorubicin or more. Blockade of PS with lactadherin inhibited over 90% of HUVECs and EMPs PCA. However, anti-TF antibody had no significant inhibition effect. Our results demonstrate that daunorubicin treatment enhanced PCA of HUVECs through PS exposure and shedding of procoagulant EMPs. Lactadherin acts as an efficient anticoagulant in this process. more...
- Published
- 2010
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9. Phosphatidylserine exposure and procoagulant activity in acute promyelocytic leukemia.
- Author
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Zhou J, Shi J, Hou J, Cao F, Zhang Y, Rasmussen JT, Heegaard CW, and Gilbert GE
- Subjects
- Adolescent, Adult, Antineoplastic Agents pharmacology, Arsenic Trioxide, Arsenicals pharmacology, Blood Coagulation Tests, Cell Differentiation drug effects, Cell Line, Tumor, Cell Survival drug effects, Daunorubicin pharmacology, Etoposide pharmacology, Factor Xa metabolism, Female, Flow Cytometry, Humans, Leukemia, Promyelocytic, Acute pathology, Male, Membrane Glycoproteins metabolism, Microscopy, Confocal, Milk Proteins metabolism, Oxides pharmacology, Thrombin metabolism, Thromboplastin metabolism, Tretinoin pharmacology, Young Adult, Blood Coagulation drug effects, Cell Membrane metabolism, Leukemia, Promyelocytic, Acute blood, Phosphatidylserines metabolism
- Abstract
Background: Acute promyelocytic leukemia (APL) frequently causes disseminated intravascular coagulation that can worsen with cytotoxic chemotherapy but improve with the therapeutic differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)). APL cells display tissue factor but the relationship of tissue factor and other procoagulant activity to phosphatidylserine (PS) exposure is largely unknown., Methods: Lactadherin, a milk protein with stereospecific binding to phosphatidyl-L-serine, was used as a probe for PS exposure on an immortalized APL cell line (NB4) and on the cells of eight patients with APL. PS exposure was evaluated with flow cytometry, confocal microscopy, coagulation assays, and purified prothrombinase and factor (F) Xase assays., Results: Plasma procoagulant activity of NB4 and APL cells increased approximately 15-fold after exposure to etoposide or daunorubicin and decreased 80% after treatment with ATRA or As(2)O(3). Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure decreased after treatment with ATRA or As(2)O(3) and increased after treatment with daunorubicin or etoposide. Excess lactadherin inhibited 80-85% of intrinsic FXase, FVIIa-tissue factor and prothrombinase activities on both NB4 cells and APL cells. Confocal microscopy identified membrane patches that stained with lactadherin, but not annexin V, demonstrating focal, low-level PS exposure., Conclusions: PS is exposed on viable APL cells and is necessary for approximately 80% of procoagulant activity. more...
- Published
- 2010
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10. Daunorubicin induces procoagulant response through phosphatidylserine exposure in red blood cells.
- Author
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Zhou J, Zheng Y, Shi J, Lu C, Hou J, Yu H, Qiao X, Qi S, and Gilbert GE
- Subjects
- Blood Coagulation physiology, Case-Control Studies, Dose-Response Relationship, Drug, Erythrocytes metabolism, Humans, Leukemia, Myeloid, Acute metabolism, Time Factors, Antineoplastic Agents pharmacology, Blood Coagulation drug effects, Daunorubicin pharmacology, Erythrocytes drug effects, Phosphatidylserines metabolism
- Abstract
Introduction: Cytotoxic chemotherapy induces or worsens hemostatic disorders in patients with acute myeloid leukemia. Procoagulant release and tissue factor expression on tumor cells are considered to contribute to chemotherapeutic agents-associated coagulopathy. The role of red blood cells during this process has not been determined; although they lack tissue factor, they may contribute suitable membranes for the amplification phase of blood coagulation. The present study aims to evaluate the possible impact of daunorubicin on phosphatidylserine exposure and consequent procoagulant property of red blood cells., Materials and Methods: Red blood cells from acute myeloid leukemia patients and healthy donors were treated with daunorubicin as well as all-trans-retinoic acid, arsenic trioxide or etoposide for 0-48 h. Procoagulant activity was assessed by measurement of a clotting time and by purified coagulation complex assays. Lactadherin was used as a probe for phosphatidylserine., Results: Daunorubicin treatment increased procoagulant activity of red blood cells regardless of the cell origin. Moreover, coagulation complexes assays supported daunorubicin-evoked procoagulant response. The procoagulant property of red blood cells was not affected by the other three agents. The modulating effect of procoagulant activity was concomitant with and dependent on level of phosphatidylserine on the outer surface. Blockade of phosphatidylserine with lactadherin inhibited over 90% of tenase generation and prothrombinase activity and prolonged the coagulation time., Conclusions: We conclude that daunorubicin interacts with red blood cells in a manner that increases phosphatidylserine exposure and consequent procoagulant activity. Lactadherin is an efficient anticoagulant of this process., (Copyright 2009 Elsevier Ltd. All rights reserved.) more...
- Published
- 2010
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11. Bovine lactadherin as a calcium-independent imaging agent of phosphatidylserine expressed on the surface of apoptotic HeLa cells.
- Author
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Waehrens LN, Heegaard CW, Gilbert GE, and Rasmussen JT
- Subjects
- Animals, Annexin A5 metabolism, Cattle, Fluorescent Dyes, HeLa Cells, Humans, Microscopy, Confocal, Staurosporine pharmacology, Apoptosis, Calcium metabolism, Membrane Glycoproteins metabolism, Milk Proteins metabolism, Phosphatidylserines biosynthesis
- Abstract
Bovine lactadherin holds a stereo-specific affinity for phosphatidylserine (PS) membrane domains and binds at PS concentrations lower than the benchmark PS probe, annexin V. Accordingly, lactadherin has recognized PS exposure on preapoptotic immortalized leukemia cells at an earlier time point than has annexin V. In the present study, the cervical cancer cell line HeLa has been employed as a model system to compare the topographic distribution of PS with the two PS binding proteins as adherent cells enter the apoptotic program. HeLa cells were cultured on glass-bottom Petri dishes, and apoptosis was induced by staurosporine. Fluorescence-labeled lactadherin and/or annexin V were used to detect PS exposure by confocal microscopy. Both lactadherin and annexin V staining revealed PS localized to plasma membrane rim and blebs. In addition, lactadherin identified PS exposure on long filopodia-like extensions, whereas annexin V internalized in granule-like structures. All in all, the data further delineate the differences in PS binding patterns of lactadherin and annexin V. more...
- Published
- 2009
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12. Procoagulant activity and phosphatidylserine of amniotic fluid cells.
- Author
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Zhou J, Liu S, Ma M, Hou J, Yu H, Lu C, Gilbert GE, and Shi J
- Subjects
- Adult, Amniotic Fluid cytology, Amniotic Fluid enzymology, Blood Coagulation Tests, Factor Xa metabolism, Female, Flow Cytometry, Humans, Microscopy, Confocal, Pregnancy, Prothrombin metabolism, Amniotic Fluid metabolism, Blood Coagulation, Disseminated Intravascular Coagulation blood, Embolism, Amniotic Fluid blood, Phosphatidylserines metabolism, Thromboplastin metabolism
- Abstract
Amniotic fluid (AF) may induce disseminated intravascular coagulation (DIC) when it enters maternal circulation by breaching the placental-maternal circulation barrier. The precise mechanism of the procoagulant activity of AF is unclear, but tissue factor (TF) has been proposed to be the main cause. As one constituent of AF, AF cells accumulate and undergo apoptosis continuously. Therefore, we speculate that AF cells have procoagulant activity due to the externalisation of phosphatidylserine (PS). The present study aims to demonstrate that, in addition to TF, the PS that is externalised on AF cells is important for the procoagulant activity of AF. Ten AF samples from parturient women were analysed using lactadherin as the probe for PS. Anti-TF antibody also was used to identify TF and its associated coagulation functions in AF cells. Normal platelets, neutrophils, and lymphocytes were harvested as controls. Confocal microscopy and flow cytometry was used to assess PS expression on AF cells. The procoagulant activity of AF cells was demonstrated by a plasma coagulation assay and further confirmed by factor Xase/prothrombinase assays. PS and TF were present on most AF cells, providing substantial procoagulant activity. Furthermore, factor Xase and prothrombinase assays showed that AF cells substantially enforced the activation of factor X and prothrombin. PS on AF cells is an important procoagulant source for AF. Lactadherin is an ideal anticoagulant for inhibiting the procoagulant activity of AF cells. more...
- Published
- 2009
13. Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure.
- Author
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Shi J, Pipe SW, Rasmussen JT, Heegaard CW, and Gilbert GE
- Subjects
- Animals, Annexin A5, Binding Sites, Blood Platelets chemistry, Carotid Artery Diseases, Cysteine Endopeptidases, Mesenteric Veins, Mice, Neoplasm Proteins antagonists & inhibitors, Thromboplastin antagonists & inhibitors, Antigens, Surface pharmacology, Blood Platelets physiology, Hemostasis drug effects, Milk Proteins pharmacology, Phosphatidylserines metabolism, Thrombosis prevention & control
- Abstract
Background: Platelet membrane phosphatidylserine (PS) is considered to be essential for hemostasis and thrombosis, but the in vivo topography of platelet PS has not been characterized. We hypothesized that platelet PS exposure would be identified on adherent platelets at the site of vascular injury and that blockade of PS would impede hemostasis and thrombosis., Objective: To localize and estimate the extent of platelet PS exposure and evaluate the impact of PS blockade in vivo., Methods: Lactadherin, a PS-binding milk protein, was utilized together with annexin V to detect both partial and complete membrane PS exposure on platelets in a mouse model of thrombosis and to evaluate the functional need for PS. Preliminary experiments were performed with synthetic membranes and with purified platelets., Results: The number of lactadherin-binding sites on synthetic membranes was proportional to PS content, whereas annexin V required a threshold of 2.5-8% PS. Approximately 95% of thrombin-stimulated platelets exposed PS, but the quantity was below the threshold for annexin V binding at physiologic Ca(2+) concentrations. In mice, most adherent and aggregated platelets on the walls of ferric chloride-treated mesenteric veins exposed low levels of PS, rather than having complete exposure. In mice, blockade of PS with lactadherin inhibited platelet prothrombinase and factor Xase activity, and prolonged tail bleeding time and the time to carotid artery thrombosis., Conclusions: In vivo PS exposure contributes to both hemostasis and thrombosis. In this model of vascular injury, most platelets exhibit partial rather than complete PS exposure. more...
- Published
- 2008
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14. Membrane phosphatidylserine regulates surface charge and protein localization.
- Author
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Yeung T, Gilbert GE, Shi J, Silvius J, Kapus A, and Grinstein S
- Subjects
- Animals, Biosensing Techniques, Cell Line, Endocytosis, Fluorescence, Fluorescence Resonance Energy Transfer, Humans, Hydrophobic and Hydrophilic Interactions, Microscopy, Confocal, Milk Proteins metabolism, Organelles metabolism, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae metabolism, Signal Transduction, Static Electricity, Surface Properties, Cell Membrane metabolism, Endosomes metabolism, Intracellular Membranes metabolism, Lysosomes metabolism, Phosphatidylserines metabolism
- Abstract
Electrostatic interactions with negatively charged membranes contribute to the subcellular targeting of proteins with polybasic clusters or cationic domains. Although the anionic phospholipid phosphatidylserine is comparatively abundant, its contribution to the surface charge of individual cellular membranes is unknown, partly because of the lack of reagents to analyze its distribution in intact cells. We developed a biosensor to study the subcellular distribution of phosphatidylserine and found that it binds the cytosolic leaflets of the plasma membrane, as well as endosomes and lysosomes. The negative charge associated with the presence of phosphatidylserine directed proteins with moderately positive charge to the endocytic pathway. More strongly cationic proteins, normally associated with the plasma membrane, relocalized to endocytic compartments when the plasma membrane surface charge decreased on calcium influx. more...
- Published
- 2008
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15. Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.
- Author
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Shi J, Shi Y, Waehrens LN, Rasmussen JT, Heegaard CW, and Gilbert GE
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- Annexin A5 pharmacokinetics, Annexin A5 pharmacology, Apoptosis, Etoposide, Fluorescent Dyes pharmacology, HL-60 Cells, Humans, K562 Cells, Milk Proteins pharmacokinetics, Antigens, Surface pharmacology, Flow Cytometry methods, Leukemia, Erythroblastic, Acute metabolism, Leukemia, Promyelocytic, Acute metabolism, Microscopy, Confocal methods, Milk Proteins pharmacology, Phosphatidylserines metabolism
- Abstract
Background: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins., Methods: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis., Results: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V., Conclusions: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology. more...
- Published
- 2006
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16. Lysine 5 and phenylalanine 9 of the factor IX omega-loop interact with phosphatidylserine in a membrane-mimetic environment.
- Author
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Grant MA, Baikeev RF, Gilbert GE, and Rigby AC
- Subjects
- Calcium chemistry, Cations, Divalent chemistry, Circular Dichroism, Factor IX genetics, Hemophilia B genetics, Humans, Lysine genetics, Models, Molecular, Molecular Mimicry, Nuclear Magnetic Resonance, Biomolecular, Peptides, Cyclic chemical synthesis, Peptides, Cyclic metabolism, Phenylalanine genetics, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Factor IX chemistry, Factor IX metabolism, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Lysine metabolism, Phenylalanine metabolism, Phosphatidylserines metabolism
- Abstract
The binding of factor IX to cell membranes requires a structured N-terminal omega-loop conformation that exposes hydrophobic residues for a highly regulated interaction with a phospholipid. We hypothesized that a peptide comprised of amino acids Gly4-Gln11 of factor IX (fIX(G4)(-)(Q11)) and constrained by an engineered disulfide bond would assume the native factor IX omega-loop conformation in the absence of Ca(2+). The small size and freedom from aggregation-inducing calcium interactions would make fIX(G4)(-)(Q11) suitable for structural studies for eliciting details about phospholipid interactions. fIX(G4)(-)(Q11) competes with factor IXa for binding sites on phosphatidylserine-containing membranes with a K(i) of 11 microM and inhibits the activation of factor X by the factor VIIIa-IXa complex with a K(i) of 285 microM. The NMR structure of fIX(G4)(-)(Q11) reveals an omega-loop backbone fold and side chain orientation similar to those found in the calcium-bound factor IX Gla domain, FIX(1-47)-Ca(2+). Dicaproylphosphatidylserine (C(6)PS) induces HN, Halpha backbone, and Hbeta chemical shift perturbations at residues Lys5, Leu6, Phe9, and Val10 of fIX(G4)(-)(Q11), while selectively protecting the NHzeta side chain resonance of Lys5 from solvent exchange. NOEs between the aromatic ring protons of Phe9 and specific acyl chain protons of C(6)PS indicate that these phosphatidylserine protons reside 3-6 A from Phe9. Stabilization of the phosphoserine headgroup and glycerol backbone of C(6)PS identifies that phosphatidylserine is in a protected environment that is spatially juxtaposed with fIX(G4)(-)(Q11). Together, these data demonstrate that Lys5, Leu6, Phe9, and Val10 preferentially interact with C(6)PS and allow us to correlate known hemophilia B mutations of factor IX at Lys5 or Phe9 with impaired phosphatidylserine interaction. more...
- Published
- 2004
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17. Lactadherin binds selectively to membranes containing phosphatidyl-L-serine and increased curvature.
- Author
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Shi J, Heegaard CW, Rasmussen JT, and Gilbert GE
- Subjects
- Animals, Antigens, Surface metabolism, Binding Sites, Cattle, Cell Shape, Factor V, Factor VIII, Membrane Lipids metabolism, Milk Proteins metabolism, Phosphatidylcholines, Phosphatidylethanolamines, Phosphatidylserines metabolism, Protein Binding, Antigens, Surface chemistry, Membrane Lipids chemistry, Milk Proteins chemistry, Phosphatidylserines chemistry
- Abstract
Lactadherin, a milk protein, contains discoidin-type lectin domains with homology to the phosphatidylserine-binding domains of blood coagulation factor VIII and factor V. We have found that lactadherin functions, in vitro, as a potent anticoagulant by competing with blood coagulation proteins for phospholipid binding sites [J. Shi and G.E. Gilbert, Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites, Blood 101 (2003) 2628-2636]. We wished to characterize the membrane-binding properties that correlate to the anticoagulant capacity. We labeled bovine lactadherin with fluorescein and evaluated binding to membranes of composition phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine, 4:20:76 supported by 2 mum diameter glass microspheres. Lactadherin bound saturably with an apparent KD of 3.3+/-0.4 nM in a Ca++ -independent manner. The number of lactadherin binding sites increased proportionally to the phosphatidylserine content over a range 0-2% and less rapidly for higher phosphatidylserine content. Inclusion of phosphatidylethanolamine in phospholipid vesicles did not enhance the apparent affinity or number of lactadherin binding sites. The number of sites was at least 4-fold higher on small unilamellar vesicles than on large unilamellar vesicles, indicating that lactadherin binding is enhanced by membrane curvature. Lactadherin bound to membranes with synthetic dioleoyl phosphatidyl-L-serine but not dioleoyl phosphatidyl-D-serine indicating stereoselective recognition of phosphatidyl-L-serine. We conclude that lactadherin resembles factor VIII and V with stereoselective preference for phosphatidyl-L-serine and preference for highly curved membranes. more...
- Published
- 2004
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18. Partial activation of the factor VIIIa-factor IXa enzyme complex by dihexanoic phosphatidylserine at submicellar concentrations.
- Author
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Gilbert GE and Arena AA
- Subjects
- Calcium pharmacology, Enzyme Activation drug effects, Factor IXa chemistry, Factor VIIIa chemistry, Humans, Lipid Bilayers metabolism, Macromolecular Substances, Micelles, Phosphatidic Acids pharmacology, Phosphatidylethanolamines pharmacology, Phosphatidylglycerols pharmacology, Phosphatidylserines chemistry, Phosphatidylserines metabolism, Factor IXa metabolism, Factor VIIIa metabolism, Phosphatidylserines pharmacology
- Abstract
Phosphatidylserine (PS)-containing membranes increase the kcat of the factor VIIIa-factor IXa enzyme complex by more than 1000-fold. While PS supports specific, high-affinity membrane binding of factor VIIIa and factor IXa, it is not known whether PS is the lipid that activates the membrane-bound complex. It is also not known whether PS or other activating lipids must reside in the two-dimensional membrane matrix for efficacy. We have found that submicellar concentrations of dihexanoic phosphatidylserine (C6PS) increase the activity of the factor VIIIa-factor IXa complex in a biphasic manner with half-maximal concentrations of 0.2 and 1.6 mM while the micelle-forming concentration is 4.0 mM. Increased cleavage of factor X at 0.25 mM C6PS was due to a 25-fold enhancement of the kcat and a 30-fold increase in the affinity of factor VIIIa for factor IXa. C6 phosphatidylethanolamine and C6 phosphatidic acid, but not C6 phosphatidylcholine, also accelerated the Xase complex, indicating that kcat enhancement has less structural specificity than membrane binding. Submicellar C6PS enhanced activity of factor IXa in the absence of factor VIIIa, but the effect was due to a decreased KM rather than an increased kcat. These results suggest that activation of the factor VIIIa-factor IXa complex can result from binding of individual C6PS molecules or small aggregates in the absence of a membrane bilayer. They provide a model system in which the phospholipid-induced activation may be distinguished from membrane-binding of the enzyme complex. more...
- Published
- 1997
- Full Text
- View/download PDF
19. Activation of the factor VIIIa-factor IXa enzyme complex of blood coagulation by membranes containing phosphatidyl-L-serine.
- Author
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Gilbert GE and Arena AA
- Subjects
- Animals, Brain, Cattle, Enzyme Activation, Humans, Kinetics, Liposomes, Recombinant Proteins metabolism, Thrombin metabolism, Factor IXa metabolism, Factor VIIIa metabolism, Phosphatidylserines pharmacology
- Abstract
Factor IXa, a serine protease of blood coagulation, functions at least 100,000 times more efficiently when bound to factor VIIIa on a phospholipid membrane than when free in solution. We have utilized the catalytic activity of the factor VIIIa-factor IXa complex to report the effect of phospholipid membranes on binding of factor IXa to factor VIIIa and on enzymatic cleavage of the product. The apparent affinity of factor IXa for factor VIIIa was 10-fold lower in the absence of phospholipid membranes with a KD of 46 nM versus 4.3 nM with phospholipid membranes. The Km for activation of factor X by the factor VIIIa-factor IXa complex was 1700 nM in solution, 70-fold higher than the value of 28 nM when bound to membranes containing phosphatidyl-L-serine, phosphatidylethanolamine, and phosphatidylcholine at a ratio of 4:20:76. The largest effect of phosphatidyl-L-serine-containing membranes on the factor VIIIa-factor IXa complex was the accelerated rate of peptide bond cleavage, with the k(cat) increased by 1,500-fold from 0.022 to 33 min-1. Membranes in which phosphatidyl-L-serine was replaced by phosphatidyl-D-serine, phosphatidic acid, or phosphatidylglycerol were at least 10-fold less effective for enhancing the k(cat). Thus, while membranes containing phosphatidyl-L-serine enhance condensation of the enzyme with its cofactor and substrate, their largest effect is activation of the assembled factor VIIIa-factor IXa enzyme complex. more...
- Published
- 1996
- Full Text
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20. Phosphatidylethanolamine induces high affinity binding sites for factor VIII on membranes containing phosphatidyl-L-serine.
- Author
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Gilbert GE and Arena AA
- Subjects
- Binding Sites, Blood Platelets metabolism, Flow Cytometry, Fluorescent Dyes, Humans, Recombinant Proteins metabolism, Stereoisomerism, Structure-Activity Relationship, Factor VIII metabolism, Membranes, Artificial, Phosphatidylethanolamines, Phosphatidylserines chemistry
- Abstract
Synthetic membranes of phosphatidylcholine require inclusion of at least 5% phosphatidylserine (Ptd-L-Ser) to form binding sites for factor VIII. The relatively high requirement for Ptd-L-Ser suggests that stimulated platelets may contain another membrane constituent that enhances expression of factor VIII-binding sites. We report that phosphatidylethanolamine (PE), which is exposed in concert with Ptd-L-Ser in the course of platelet stimulation, induces high affinity binding sites for factor VIII on synthetic membranes containing 1-15% Ptd-L-Ser. The affinity of factor VIII for binding sites on membranes of Ptd-L-Ser/PE/phosphatidylcholine in a 4:20:76 ratio was 10.2 +/- 3.5 nM with 180 +/- 33 phospholipid molecules/site. PE did not induce binding sites on membranes of 4% Ptd-D-Ser, indicating that the induced binding sites require the correct stereochemistry of Ptd-L-Ser as well as PE. Egg PE and dimyristoyl-PE were equivalent for inducing factor VIII-binding sites, indicating that hexagonal phase-inducing properties of PE are not important. We conclude that PE induces high affinity factor VIII-binding sites on membranes with physiologic mole fractions of Ptd-L-Ser, possibly including those of stimulated platelets. more...
- Published
- 1995
- Full Text
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21. Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine.
- Author
-
Gilbert GE and Drinkwater D
- Subjects
- Binding Sites, Blood Platelets metabolism, Humans, In Vitro Techniques, Membrane Lipids chemistry, Membrane Lipids metabolism, Membrane Potentials, Phosphatidylserines chemistry, Phosphoserine chemistry, Recombinant Proteins metabolism, Factor VIII metabolism, Phosphatidylserines metabolism, Phosphoserine metabolism
- Abstract
Phosphatidylserine, a negatively charged lipid, is exposed on the platelet membrane following cell stimulation, correlating with the expression of factor VIII receptors. We have explored the importance of the negative electrostatic potential of phosphatidylserine vs chemical moieties of phosphatidylserine for specific membrane binding of factor VIII. Fluorescein-labeled factor VIII bound to membranes containing 15% phosphatidic acid, a negatively charged phospholipid, with low affinity compared to phosphatidylserine-containing membranes. Binding was not specific as it was inhibited by other proteins in plasma. Factor VIII bound to membranes containing 10% phosphatidylserine in spite of a varying net charge provided by 0-15% stearylamine, a positively charged lipid. The soluble phosphatidylserine moiety, O-phospho-L-serine, inhibited factor VIII binding to phosphatidylserine-containing membranes with a Ki of 20 mM, but the stereoisomer, O-phospho-D-serine, was 5-fold less effective. Furthermore, binding of factor VIII to membranes containing synthetic phosphatidyl-D-serine was 5-fold less than binding to membranes containing phosphatidyl-L-serine. Membranes containing synthetic phosphatidyl-L-homoserine, differing from phosphatidylserine by a single methylene, supported high-affinity binding, but it was not specific as factor VIII was displaced by other plasma proteins. O-Phospho-L-serine also inhibited the binding of factor VIII to platelet-derived microparticles with a Ki of 20 mM, and the stereoisomer was 4-fold less effective. These results indicate that membrane binding of factor VIII is mediated by a stereoselective recognition O-phospho-L-serine of phosphatidylserine and that negative electrostatic potential is of lesser importance. more...
- Published
- 1993
- Full Text
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22. Specificity of phosphatidylserine-containing membrane binding sites for factor VIII. Studies with model membranes supported by glass microspheres (lipospheres).
- Author
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Gilbert GE, Drinkwater D, Barter S, and Clouse SB
- Subjects
- Binding Sites, Factor V metabolism, Glass, Humans, Kinetics, Light, Liposomes, Mathematics, Microscopy, Fluorescence, Microspheres, Models, Biological, Scattering, Radiation, Factor VIII metabolism, Membrane Lipids metabolism, Phosphatidylserines metabolism
- Abstract
Factor VIII functions in an enzyme complex upon the activated platelet membrane where phosphatidylserine exposure correlates with expression of receptors for factor VIII. To evaluate the specificity of phosphatidylserine-containing membrane binding sites for factor VIII, we have developed a novel membrane model in which phospholipid bilayers are supported by glass microspheres (lipospheres). The binding of fluorescein-labeled factor VIII to lipospheres with membranes of 15% phosphatidylserine was equivalent to binding to phospholipid vesicles (KD = 4.8 nM). Purified von Willebrand factor (vWf), a carrier protein for factor VIII, decreased membrane binding of factor VIII with a Ki of 10 micrograms/ml. Likewise, normal plasma decreased bound factor VIII by more than 90% whereas plasma lacking vWf decreased the binding of factor VIII by only 20%. Proteolytic activation of factor VIII by thrombin, which releases factor VIII from vWf, increased liposphere binding in the presence of vWf and in the presence of normal plasma. Although factor V is homologous to factor VIII and binds to lipospheres with the same affinity, purified factor V was not an efficient competitor for the membrane binding sites of factor VIII. These results indicate that phosphatidylserine-containing membrane sites have sufficient specificity to select thrombin-activated factor VIII from the range of phospholipid-binding proteins in plasma. more...
- Published
- 1992
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