scholarly journals Calpain cleaves phospholipid flippase ATP8A1 during apoptosis in platelets

2019 ◽  
Vol 3 (3) ◽  
pp. 219-229 ◽  
Author(s):  
Weidong Jing ◽  
Mehmet Yabas ◽  
Angelika Bröer ◽  
Lucy Coupland ◽  
Elizabeth E. Gardiner ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cysteine Protease ◽  
Calcium Influx ◽  
Human Platelets ◽  
Asymmetric Distribution ◽  
Resting Cells ◽  
Activated Platelets ◽  
Caspase Inhibition ◽  
Phospholipid Flippase

Abstract The asymmetric distribution of phospholipids in the plasma/organellar membranes is generated and maintained through phospholipid flippases in resting cells, but becomes disrupted in apoptotic cells and activated platelets, resulting in phosphatidylserine (PS) exposure on the cell surface. Stable PS exposure during apoptosis requires inactivation of flippases to prevent PS from being reinternalized. Here we show that flippase ATP8A1 is highly expressed in both murine and human platelets, but is not present in the plasma membrane. ATP8A1 is cleaved by the cysteine protease calpain during apoptosis, and the cleavage is prevented indirectly by caspase inhibition, involving blockage of calcium influx into platelets and subsequent calpain activation. In contrast, in platelets activated with thrombin and collagen and exposing PS, ATP8A1 remains intact. These data reveal a novel mechanism of flippase cleavage and suggest that flippase activity in intracellular membranes differs between platelets undergoing apoptosis and activation.

scholarly journals Crystal structure of a human plasma membrane phospholipid flippase

2019 ◽  
Author(s):  
Hanayo Nakanishi ◽  
Katsumasa Irie ◽  
Katsumori Segawa ◽  
Kazuya Hasegawa ◽  
Yoshinori Fujiyoshi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Human Plasma ◽  
Binding Sites ◽  
Living Cell ◽  
Asymmetric Distribution ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Flippase ◽  
Plasma Membrane Phospholipid

AbstractATP11C, a member of P4-ATPase flippase, exclusively translocates phosphatidylserine from the outer to the inner leaflets of the plasma membrane, and maintains the asymmetric distribution of phosphatidylserine in the living cell. However, the mechanisms by which ATP11C translocates phosphatidylserine remain elusive. Here we show the crystal structures of a human plasma membrane flippase, ATP11C-CDC50A complex, in an outward-open E2P conformation. Two phosphatidylserine molecules are in a conduit that continues from the cell surface to the occlusion site in the middle of the membrane. Mutations in either of the phosphotidylserine binding sites or along the pathway between significantly impairs specific ATPase and transport activities. We propose a model for phosphatidylserine translocation from the outer to the inner leaflet of the plasma membrane.

scholarly journals The secretory pathway of bovine platelets

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 878-885 ◽  
Author(s):  
JG White
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Human Platelet ◽  
Surface Membrane ◽  
Surrounding Medium ◽  
Human Platelets ◽  
Resting Cells ◽  
Granule Secretion ◽  
New Perspective ◽  
Platelet Secretion

Abstract Human platelets contain tortuous channels in their cytoplasm, the surface-connected or open canalicular system (OCS), that communicate directly with the surrounding medium through openings on the surface membrane. Some workers have suggested that the OCS serves as the egress route for products secreted during the release reaction. Others have proposed alternate secretory pathways. Since bovine platelets lack the OCS found in human cells, the present study has examined the secretory mechanism of these cells to see whether it can shed light on the mystery of human platelet secretion. Bovine platelet granules, in contrast to human granules, are located more peripherally in resting cells (often in contact with the plasma membrane), most do not move centrally following thrombin stimulation as do human platelet granules, and many fuse directly with the external plasma membrane without any intermediate channel. The lack of peripheral location of human granules, their central rather than peripheral movement during secretion, and the presence of extensive channels are all consistent with the larger importance of the secretory channel to human platelets. Thus, though studies of bovine secretion do show that platelets can secrete their granules by direct fusion of granule and surface membranes, other differences from human platelets emphasize that this pathway, although important to bovine platelet secretion, is less important in human platelets. Studies of bovine platelets also show that the OCS is more dynamic than might have been considered from human studies and can form rapidly in response to stimulation. Such newly formed channels are used as a conduit for secretion of granule contents. The finding emphasizes the importance of channels for granule secretion in platelets generally and puts a new perspective on the ability of these cells to form channels rapidly in response to stimulation.

scholarly journals Studies on the mechanism of expression of secreted fibrinogen on the surface of activated human platelets

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1226-1234 ◽  
Author(s):  
C Legrand ◽  
V Dubernard ◽  
AT Nurden
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Synthetic Peptides ◽  
Divalent Cations ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Plasma Fibrinogen ◽  
Fab Fragments ◽  
Polymer Formation ◽  
Activated Platelets

Abstract Affinity purified anti-fibrinogen (anti-Fg) Fab fragments were used to study the mechanism of expression of alpha-granule fibrinogen on activated platelets. Low amounts of the radiolabeled anti-Fg Fab bound to unstimulated or adenosine diphosphate (ADP)-stimulated cells. They readily bound to platelets stimulated with collagen, alpha-thrombin or gamma-thrombin in the presence of divalent cations. At 1 n mol/L alpha- thrombin or 25 nmol/L gamma-thrombin, platelet fibrinogen was expressed on the surface of the cells notwithstanding the presence of AP-2, a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, or the synthetic peptides Arg-Gly-Asp-Ser and gamma 400–411, all substances that prevented the binding of plasma fibrinogen to platelets. These results suggest that platelet fibrinogen may interact with its receptors during its translocation from the alpha-granules to the plasma membrane and, thus, not occupy the same sites as those available for plasma fibrinogen on the surface of the cell. Furthermore, we found that platelet fibrinogen was expressed on the thrombin-stimulated platelets of a Glanzmann's thrombasthenia variant that failed to bind plasma fibrinogen. Normal platelets stimulated with 5 nmol/L alpha- thrombin bound increased amounts of the anti-fg Fab, the additional expression being inhibited by the anti-GP IIb-IIIa monoclonal antibody or by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymer formation. This suggests that rebinding to externally located GP IIb-IIIa complexes becomes important once fibrin is formed.

scholarly journals Crystal structure of a human plasma membrane phospholipid flippase

2020 ◽  
Vol 295 (30) ◽  
pp. 10180-10194 ◽  
Author(s):  
Hanayo Nakanishi ◽  
Katsumasa Irie ◽  
Katsumori Segawa ◽  
Kazuya Hasegawa ◽  
Yoshinori Fujiyoshi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Human Plasma ◽  
Binding Sites ◽  
The Other ◽  
Asymmetric Distribution ◽  
Transmembrane Helices ◽  
Outer Leaflet ◽  
Extracellular Side ◽  
Phospholipid Flippase ◽  
Plasma Membrane Phospholipid

ATP11C, a member of the P4-ATPase flippase, translocates phosphatidylserine from the outer to the inner plasma membrane leaflet, and maintains the asymmetric distribution of phosphatidylserine in the living cell. We present the crystal structures of a human plasma membrane flippase, ATP11C–CDC50A complex, in a stabilized E2P conformation. The structure revealed a deep longitudinal crevice along transmembrane helices continuing from the cell surface to the phospholipid occlusion site in the middle of the membrane. We observed that the extension of the crevice on the exoplasmic side is open, and the complex is therefore in an outward-open E2P state, similar to a recently reported cryo-EM structure of yeast flippase Drs2p–Cdc50p complex. We noted extra densities, most likely bound phosphatidylserines, in the crevice and in its extension to the extracellular side. One was close to the phosphatidylserine occlusion site as previously reported for the human ATP8A1–CDC50A complex, and the other in a cavity at the surface of the exoplasmic leaflet of the bilayer. Substitutions in either of the binding sites or along the path between them impaired specific ATPase and transport activities. These results provide evidence that the observed crevice is the conduit along that phosphatidylserine traverses from the outer leaflet to its occlusion site in the membrane and suggest that the exoplasmic cavity is important for phospholipid recognition. They also yield insights into how phosphatidylserine is incorporated from the outer leaflet of the plasma membrane into the transmembrane.

scholarly journals Econazole inhibits thapsigargin-induced platelet calcium influx by mechanisms other than cytochrome P-450 inhibition

1993 ◽  
Vol 295 (2) ◽  
pp. 525-529 ◽  
Author(s):  
J G Vostal ◽  
J C Fratantoni
Keyword(s):  
Plasma Membrane ◽  
Antifungal Agents ◽  
Calcium Influx ◽  
Calcium Stores ◽  
Alternative Mechanism ◽  
Resting Cells ◽  
Calcium Efflux ◽  
Plasma Membrane Permeability ◽  
Cytochrome P 450 ◽  
Internal Calcium

Cytochrome P-450 has been suggested as a mediator of the signal between depleted platelet calcium stores and an increase in plasma membrane permeability to calcium which follows depletion of the stores. This hypothesis is based on the observations that inhibitors of cytochrome P-450, such as the imidazole antifungal agents, also inhibit influx of a calcium surrogate (manganese) into calcium-depleted platelets. We tested the effects of econazole and of a cytochrome P-450 inhibitor, carbon monoxide (CO), on thapsigargin (TG)-induced platelet 45Ca2+ influx. TG specifically depletes internal calcium stores and activates store-regulated calcium influx. Econazole blocked 45Ca2+ influx when it was added before TG (IC50 11 microM). Econazole at a concentration (20 microM) that inhibited 83% of TG-induced calcium influx was not inhibitory to TG-induced calcium efflux from 45Ca(2+)-loaded platelets, and did not affect calcium fluxes in resting platelets. This econazole concentration was also inhibitory to calcium influx even when it was added after the stores had been calcium-depleted by EGTA and TG for 15 min and the signal to increase calcium influx had already been generated. Inhibition of cytochrome P-450 with CO bubbled through platelet suspensions did not change calcium influx in resting cells and potentiated TG-induced calcium influx (160% of control calcium accumulation at 20 min). This effect appeared to be concentration-dependent, such that a 5 min exposure to CO produced a greater influx potentiation than a 3 min exposure. These observations indicate that (1) cytochrome P-450 does not mediate store-regulated calcium influx, and (2) econazole probably inhibits store-regulated calcium influx by an alternative mechanism, such as interaction with plasma membrane calcium channels.

scholarly journals Molecular mechanism of Fast Endophilin-Mediated Endocytosis

2020 ◽  
Vol 477 (12) ◽  
pp. 2327-2345 ◽  
Author(s):  
Alessandra Casamento ◽  
Emmanuel Boucrot
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Molecular Mechanism ◽  
Receptor Activation ◽  
Membrane Curvature ◽  
Surface Proteins ◽  
Resting Cells ◽  
Cellular Functions ◽  
Surface Receptors ◽  
Key Steps

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Clathrin-mediated endocytosis (CME) is the housekeeping pathway in resting cells but additional Clathrin-independent endocytic (CIE) routes, including Fast Endophilin-Mediated Endocytosis (FEME), internalize specific cargoes and support diverse cellular functions. FEME is part of the Dynamin-dependent subgroup of CIE pathways. Here, we review our current understanding of the molecular mechanism of FEME. Key steps are: (i) priming, (ii) cargo selection, (iii) membrane curvature and carrier formation, (iv) membrane scission and (v) cytosolic transport. All steps are controlled by regulatory mechanisms mediated by phosphoinositides and by kinases such as Src, LRRK2, Cdk5 and GSK3β. A key feature of FEME is that it is not constitutively active but triggered upon the stimulation of selected cell surface receptors by their ligands. In resting cells, there is a priming cycle that concentrates Endophilin into clusters on discrete locations of the plasma membrane. In the absence of receptor activation, the patches quickly abort and new cycles are initiated nearby, constantly priming the plasma membrane for FEME. Upon activation, receptors are swiftly sorted into pre-existing Endophilin clusters, which then bud to form FEME carriers within 10 s. We summarize the hallmarks of FEME and the techniques and assays required to identify it. Next, we review similarities and differences with other CIE pathways and proposed cargoes that may use FEME to enter cells. Finally, we submit pending questions and future milestones and discuss the exciting perspectives that targeting FEME may boost treatments against cancer and neurodegenerative diseases.

scholarly journals The fate of the open canalicular system in surface and suspension- activated platelets

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 1983-1988 ◽  
Author(s):  
G Escolar ◽  
E Leistikow ◽  
JG White
Keyword(s):  
Cell Surface ◽  
Platelet Activation ◽  
Surface Activation ◽  
Resting Cells ◽  
Platelet Surface ◽  
Early Stages ◽  
Activated Platelets ◽  
Surface Contact ◽  
Platelet Spreading ◽  
Membrane Reservoir

Abstract We have examined the movement of fibrinogen-gold (fgn-Au) complexes in platelets activated in suspension and by surface contact. Fgn-Au probes did not react with resting cells but were bound to the external membrane of platelets in suspension 5 seconds after addition of 1 U/mL of thrombin. At intervals over a period of 5 to 20 minutes, fgn-Au probes moved from the cell surface to peripheral and then deep channels of the open canalicular system (OCS). When platelets were surface activated by exposure to carbon-stabilized, formvar-coated grids for 5 to 20 minutes and then exposed to fgn-Au complexes for 5 minutes, probes were also observed in the OCS. At 5 minutes, over 40% of the platelets had concentrated fgn-Au in their OCS. Results after 10 minutes revealed 25% with gold-filled channels, 16% after 15 minutes, and 5% after 20 minutes. The decrease in frequency of OCS staining correlated with the increasing frequency of spread platelets, suggesting that tension produced by spreading may cause collapse of the OCS or that the OCS may evaginate onto the platelet during spreading. To evaluate the latter hypothesis, platelets were initially exposed to grids for 5 minutes and then incubated with fgn-Au for intervals of 5 to 20 minutes. The frequency of platelets with fgn-Au concentrated in the OCS was greatest at 5 minutes (44%) and decreased at the same rate as the frequency of spread platelets increased. Only 14.7% of the cells contained fgn-Au in the OCS after 20 minutes. These were primarily dendritic in form, while fully spread platelets rarely contained an OCS filled with the probe. The study indicates that fgn-Au particles are cleared to channels of the OCS independent of the mechanism of platelet activation. Fgn-Au that has been concentrated in the OCS at early stages of surface activation can be externalized during platelet spreading but remain internalized in suspension-activated cells. The OCS represents a membrane reservoir that can be evaginated onto the platelet surface during interaction with surfaces.

scholarly journals Increasing the expression of calcium-permeable TRPC3 and TRPC7 channels enhances constitutive secretion

2008 ◽  
Vol 413 (3) ◽  
pp. 437-446 ◽  
Author(s):  
Verna Lavender ◽  
Setareh Chong ◽  
Katherine Ralphs ◽  
Adrian J. Wolstenholme ◽  
Barbara J. Reaves
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Transient Receptor Potential ◽  
Calcium Influx ◽  
Brefeldin A ◽  
Receptor Potential ◽  
Fold Increase ◽  
Extracellular Medium ◽  
Additional Function ◽  
Golgi Stack

The hTRPC [human TRPC (canonical transient receptor potential)] family of non-selective cation channels is proposed to mediate calcium influx across the plasma membrane via PLC (phospholipase C)-coupled receptors. Heterologously expressed hTRPC3 and hTRPC7 have been localized at the cell surface; however, a large intracellular component has also been noted but not characterized. In the present study, we have investigated the intracellular pool in COS-7 cells and have shown co-localization with markers for both the TGN (trans-Golgi network) and the cis-Golgi cisternae by immunofluorescence microscopy. Addition of BFA (Brefeldin A) to cells expressing hTRPC3 or hTRPC7 resulted in the redistribution of the Golgi component to the endoplasmic reticulum, indicating that this pool is present in both the Golgi stack and the TGN. Expression of either TRPC3 or TRPC7, but not TRPC1 or the cell surface marker CD8, resulted in a 2–4-fold increase in secreted alkaline phosphatase in the extracellular medium. Based on these results, we propose that an additional function of these members of the hTRPC family may be to enhance secretion either by affecting transport through the Golgi stack or by increasing fusion at the plasma membrane.

scholarly journals Studies on the mechanism of expression of secreted fibrinogen on the surface of activated human platelets

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1226-1234
Author(s):  
C Legrand ◽  
V Dubernard ◽  
AT Nurden
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Synthetic Peptides ◽  
Divalent Cations ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Plasma Fibrinogen ◽  
Fab Fragments ◽  
Polymer Formation ◽  
Activated Platelets

Affinity purified anti-fibrinogen (anti-Fg) Fab fragments were used to study the mechanism of expression of alpha-granule fibrinogen on activated platelets. Low amounts of the radiolabeled anti-Fg Fab bound to unstimulated or adenosine diphosphate (ADP)-stimulated cells. They readily bound to platelets stimulated with collagen, alpha-thrombin or gamma-thrombin in the presence of divalent cations. At 1 n mol/L alpha- thrombin or 25 nmol/L gamma-thrombin, platelet fibrinogen was expressed on the surface of the cells notwithstanding the presence of AP-2, a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, or the synthetic peptides Arg-Gly-Asp-Ser and gamma 400–411, all substances that prevented the binding of plasma fibrinogen to platelets. These results suggest that platelet fibrinogen may interact with its receptors during its translocation from the alpha-granules to the plasma membrane and, thus, not occupy the same sites as those available for plasma fibrinogen on the surface of the cell. Furthermore, we found that platelet fibrinogen was expressed on the thrombin-stimulated platelets of a Glanzmann's thrombasthenia variant that failed to bind plasma fibrinogen. Normal platelets stimulated with 5 nmol/L alpha- thrombin bound increased amounts of the anti-fg Fab, the additional expression being inhibited by the anti-GP IIb-IIIa monoclonal antibody or by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymer formation. This suggests that rebinding to externally located GP IIb-IIIa complexes becomes important once fibrin is formed.

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