scholarly journals Ferlins and TgDOC2 in Toxoplasma Microneme, Rhoptry and Dense Granule Secretion

Life ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 217
Author(s):  
Daniel N. A. Tagoe ◽  
Allison A. Drozda ◽  
Julia A. Falco ◽  
Tyler J. Bechtel ◽  
Eranthie Weerapana ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Lipids ◽  
Dense Granule ◽  
C2 Domain ◽  
General Role ◽  
Post Translational Modifications ◽  
C2 Domains ◽  
Apicomplexan Parasites ◽  
Dense Granule Secretion ◽  
Granule Secretion

The host cell invasion process of apicomplexan parasites like Toxoplasma gondii is facilitated by sequential exocytosis of the microneme, rhoptry and dense granule organelles. Exocytosis is facilitated by a double C2 domain (DOC2) protein family. This class of C2 domains is derived from an ancestral calcium (Ca2+) binding archetype, although this feature is optional in extant C2 domains. DOC2 domains provide combinatorial power to the C2 domain, which is further enhanced in ferlins that harbor 5–7 C2 domains. Ca2+ conditionally engages the C2 domain with lipids, membranes, and/or proteins to facilitating vesicular trafficking and membrane fusion. The widely conserved T. gondii ferlins 1 (FER1) and 2 (FER2) are responsible for microneme and rhoptry exocytosis, respectively, whereas an unconventional TgDOC2 is essential for microneme exocytosis. The general role of ferlins in endolysosmal pathways is consistent with the repurposed apicomplexan endosomal pathways in lineage specific secretory organelles. Ferlins can facilitate membrane fusion without SNAREs, again pertinent to the Apicomplexa. How temporal raises in Ca2+ combined with spatiotemporally available membrane lipids and post-translational modifications mesh to facilitate sequential exocytosis events is discussed. In addition, new data on cross-talk between secretion events together with the identification of a new microneme protein, MIC21, is presented.

Differential Regulation of α-Granule and Dense Granule Secretion by an Actin Cytoskeletal Barrier.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3528-3528
Author(s):  
Robert Flaumenhaft ◽  
James R. Dilks ◽  
Nataliya Rozenvayn ◽  
Rita A. Monahan-Earley ◽  
Dian Feng ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Actin Polymerization ◽  
Dense Granule ◽  
Snare Protein ◽  
Latrunculin A ◽  
Cytochalasin E ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Cytoskeletal Disruption ◽  
Granule Release

Abstract Platelet granule secretion is an essential component of normal arterial thrombus formation. Stimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and loss of granules. These observations have lead to the supposition that cytoskeletal contraction facilitates granule secretion. Yet, the influence of the actin cytoskeleton in controlling membrane fusion events required for granule secretion remains largely unknown. Initial studies using electron microscopy revealed that the actin disrupting agents latrunculin A (4 μM) or cytochalasin E (4 μM) prevented pseudopod formation and granule centralization in platelets exposed to SFLLRN or PMA, but did not prevent degranulation. We next determined the effects of disruption of the actin cytoskeleton on α-granule secretion by monitoring P-selectin expression and β-thromboglobulin release. Incubation of platelets with either latrunculin A or cytochalasin E failed to stimulate α-granule secretion, but increased the rate of SFLLRN-induced α-granule secretion by 3.5-fold. Cytoskeletal disruption also augmented the degree of SFLLRN-induced α-granule secretion by 41±18% and reduced the amount of SFLLRN required to cause half-maximal stimulation by 2-fold. Incubation with latrunculin A stimulated α-granule secretion by the weak secretogues epinephrine or ADP by 7.6-fold and 5.4-fold, respectively. Cytoskeletal disruption also facilitated β-thromboglobulin release in response to SFLLRN, epinephrine, or ADP. In platelets permeabilized in the absence of ATP, exposure to 2 μM latrunculin A resulted in a 6.5- and 3.5-fold increase in α-granule release induced by Ca2+- or GTP-γ-S, respectively. Antibodies directed at a SNARE protein termed vesicle-associated fusion protein (VAMP) inhibited latrunculin A-dependent α-granule secretion. Thus, disruption of the actin cytoskeletal barrier by latrunculin A supports SNARE protein-dependent membrane fusion. Since actin acts as a barrier to α-granule secretion, we evaluated α-granules purified by subcellular fractionation for the presence of F-actin. Purified α-granules, but not phospholipid micelles, bound the F-actin probe FITC-phalloidin as determined by flow cytometry. FITC-phalloidin binding was inhibited in a dose-dependent manner by latrunculin A. These data indicated that α-granules are coated with F-actin that could serve a barrier function. We next evaluated the effects of cytoskeletal disruption on dense granule secretion by monitoring ADP/ATP release using a luciferin-luciferase based assay and by quantifying [3H]serotonin release. Cytoskeletal disruption by 4 μM latrunculin A failed to affect the degree of dense granule secretion from platelets stimulated by either SFLLRN, epinephrine, or ADP. Yet, 200 μM latrunculin A stimulated substantial dense granule release in the absence of agonist exposure and augmented SFLLRN-induced dense granule release by 2-fold. In contrast, 200 μM latrunculin A abolished SFLLRN-induced α-granule secretion. These observations indicate that the cytoskeleton differentially regulates α-granule and dense granule secretion. Our results also suggest that while some degree of actin polymerization is required for α-granule secretion, dense granule secretion is not dependent on actin polymerization.

The actin cytoskeleton differentially regulates platelet α-granule and dense-granule secretion

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 3879-3887 ◽  
Author(s):  
Robert Flaumenhaft ◽  
James R. Dilks ◽  
Nataliya Rozenvayn ◽  
Rita A. Monahan-Earley ◽  
Dian Feng ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Fusion ◽  
Dense Granule ◽  
Protein Receptor ◽  
Low Concentrations ◽  
Latrunculin A ◽  
High Concentrations ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Platelet Exocytosis

AbstractStimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and secretion of granules. These observations have led to the supposition that cytoskeletal contraction facilitates granule release by promoting the interaction of granules with one another and with membranes of the open canalicular system. Yet, the influence of the actin cytoskeleton in controlling the membrane fusion events that mediate granule secretion remains largely unknown. To evaluate the role of the actin cytoskeleton in platelet granule secretion, we have assessed the effects of latrunculin A and cytochalasin E on granule secretion. Exposure of platelets to low concentrations of these reagents resulted in acceleration and augmentation of agonist-induced α-granule secretion with comparatively modest effects on dense granule secretion. In contrast, exposure of platelets to high concentrations of latrunculin A inhibited agonist-induced α-granule secretion but stimulated dense granule secretion. Incubation of permeabilized platelets with low concentrations of latrunculin A primed platelets for Ca2+- or guanosine triphosphate (GTP)-γ-S-induced α-granule secretion. Latrunculin A-dependent α-granule secretion was inhibited by antibodies directed at vesicle-associated membrane protein (VAMP), demonstrating that latrunculin A supports soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent membrane fusion. These results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates α-granule and dense granule secretion.

THE ROLE OF IONISED INTRACELLULAR FREE CALCIUM ([Ca++]i) IN THROMBIN-INDUCED DENSE GRANULE SECRETION

1987 ◽  
Author(s):  
C T Poll ◽  
J Westwick
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Free Calcium ◽  
Fluorescent Properties ◽  
Fura 2 ◽  
Stimulus Response ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion

Fura 2 is one of a recently-introduced family of Ca++ indicators with improved fluorescent properties compared to quin 2 (Grynkiewicz et al 1985). This study has examined the role of [Ca++]i in thrombin-induced dense granule release using prostacyclin-washed human platelets loaded with either thedense granule marker 14C-5HT (5HT) alone or with 5HT together with quin 2 ([quin2]i = 0.8mM) or fura 2 ([fura 2]i 20-30µM). In the presence of ImM extracellular calcium concentration ([Ca++]i) the [Ca++]e in quin 2 and fura 2 loaded platelets was 93±2 (n=10 experiments) and 133±0.3nM (n=12 experiments) respectively. In either quin 2 or fura 2 loaded platelets suspended in the presence of ImM [Ca++]e, thrombin (0.23-23.InM) promoted a rapid (in secs)concentration-dependent elevation of [Ca++]i from basal values to levels l-2µM, together with a parallel release of dense granules almost identical to that obtained with thrombin in non dye loaded platelets. In fura 2 loaded cells, removal of [Ca++]e inhibited the elevation of [Ca++]i induced by a sub-maximal concentration of thrombin (0.77nM) by 43+5% (n=4) but interestingly had no significant effect (p<0.05) on the rise in [Ca++]i elicited by low thrombin doses (0.231nM). Neither did lowering [Ca++]e inhibit the release of 5HT evoked by thrombin ( 0.231-23.InM) from either fura 2 loaded or non dye loaded platelets. In contrast, in quin 2 loaded platelets, removal of [Ca++]e inhibited the thrombin (0.231-23.InM) stimulated rise in [Ca++]i-by 90% and the 5HT release response to either low (0.231nM), sub-maximal (0.77nM) or maximal (23.InM) thrombin by 100% (n=4), 87+2°/o (n=6)and 2+l°/o (n=4) respectively. Fura 2 but not quin 2 loaded cells suspended in ImM [Ca++]e exhibited a Ca++ response to thrombin concentrations >2.31nM which could be separated into a rapid phasic component and a more sustained 'tonic' like component inhibitable by removal of [Ca++]e or by addition of ImM Ni++ . These data suggest the use of fura 2 rather than quin 2 for investigating stimulus response coupling in platelets, particularly when [Ca++]e is less than physiological. We thank the British Heart Foundation and Ciba-Geigy USA for financial support.

scholarly journals Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides

1979 ◽  
Vol 182 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Holm Holmsen ◽  
Linda Robkin ◽  
H. James Day
Keyword(s):  
Shape Change ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Dense Granule ◽  
Metabolic Inhibitors ◽  
Antimycin A ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Dense Granule Secretion ◽  
Granule Secretion

1. Shape change, aggregation and secretion of dense-granule constituents in platelets differ in their dependence on cellular energy metabolism. The possibility that such a difference also exists between secretion of dense-granule constituents and acid hydrolases was investigated. 2. Human platelets were incubated with [14C]adenine in plasma, and then washed and resuspended in salt solutions. The effects of incubating the cells with antimycin A and 2-deoxyglucose on the concentrations of [14C]ATP, ADP, AMP, IMP and inosine plus hypoxanthine and on thrombin-induced secretion of ATP plus ADP and acid hydrolases were studied. The metabolic inhibitors only affected 14C-labelled nucleotides, whereas thrombin only liberated unlabelled ATP and ADP. 3. The extent of secretion decreased progressively with time during incubation with the metabolic inhibitors. At any time the secretion of acid hydrolases, β-N-acetylglucosaminidase, β-glucuronidase and β-galactosidase was inhibited to a greater extent than secretion of ATP plus ADP (dense-granule secretion). 4. Incubation with the metabolic inhibitors shifted the log (dose)–response relationship to higher thrombin concentrations, and with a greater shift for acid hydrolase secretion than for dense-granule secretion. 5. Antimycin, when present alone, caused a marked decrease in the rate of acid hydrolase secretion, but had no effect on dense-granule secretion. 6. These results further support the view that acid hydrolase secretion and dense-granule secretion are separate processes with different requirements for ATP energy. Acid hydrolase secretion, but not dense-granule secretion, appears to depend on a simultaneous rapid generation of ATP, which can be accomplished by oxidative, but not by glycolytic, ATP production.

scholarly journals Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability

2014 ◽  
Vol 290 (3) ◽  
pp. 1536-1545 ◽  
Author(s):  
Ewelina M. Golebiewska ◽  
Matthew T. Harper ◽  
Christopher M. Williams ◽  
Joshua S. Savage ◽  
Robert Goggs ◽  
...  
Keyword(s):  
Dense Granule ◽  
Dense Granule Secretion ◽  
Granule Secretion

scholarly journals Loss of the exocyst complex component EXOC3 promotes hemostasis and accelerates arterial thrombosis

2021 ◽  
Vol 5 (3) ◽  
pp. 674-686
Author(s):  
Tony G. Walsh ◽  
Yong Li ◽  
Christopher M. Williams ◽  
Elizabeth W. Aitken ◽  
Robert K. Andrews ◽  
...  
Keyword(s):  
Arterial Thrombosis ◽  
Surface Expression ◽  
Dense Granule ◽  
Conditional Knockout ◽  
Integrin Activation ◽  
Platelet Factor ◽  
Glycoprotein Vi ◽  
Exocyst Complex ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract The exocyst is an octameric complex comprising 8 distinct protein subunits, exocyst complex components (EXOC) 1 to 8. It has an established role in tethering secretory vesicles to the plasma membrane, but its relevance to platelet granule secretion and function remains to be determined. Here, EXOC3 conditional knockout (KO) mice in the megakaryocyte/platelet lineage were generated to assess exocyst function in platelets. Significant defects in platelet aggregation, integrin activation, α-granule (P-selectin and platelet factor 4), dense granule, and lysosomal granule secretion were detected in EXOC3 KO platelets after treatment with a glycoprotein VI (GPVI)-selective agonist, collagen-related peptide (CRP). Except for P-selectin exposure, these defects were completely recovered by maximal CRP concentrations. GPVI surface levels were also significantly decreased by 14.5% in KO platelets, whereas defects in proximal GPVI signaling responses, Syk and LAT phosphorylation, and calcium mobilization were also detected, implying an indirect mechanism for these recoverable defects due to decreased surface GPVI. Paradoxically, dense granule secretion, integrin activation, and changes in surface expression of integrin αIIb (CD41) were significantly increased in KO platelets after protease-activated receptor 4 activation, but calcium responses were unaltered. Elevated integrin activation responses were completely suppressed with a P2Y12 receptor antagonist, suggesting enhanced dense granule secretion of adenosine 5′-diphosphate as a critical mediator of these responses. Finally, arterial thrombosis was significantly accelerated in KO mice, which also displayed improved hemostasis determined by reduced tail bleeding times. These findings reveal a regulatory role for the exocyst in controlling critical aspects of platelet function pertinent to thrombosis and hemostasis.

REQUIREMENT FOR THROMBIN RECEPTOR OCCUPANY DURING PLATELET SECRETION UNDER AGGREGATING CONDITIONS

1987 ◽  
Author(s):  
Kazuo Koike ◽  
Holm Holmsen
Keyword(s):  
Receptor Occupancy ◽  
Dense Granule ◽  
Secretory Process ◽  
Thrombin Receptor ◽  
Cell Contact ◽  
Acid Hydrolase ◽  
High Concentration ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Platelet Secretion

We have previously showed that hirudin abruptly arrests thrombin-induced secretion of acid hydrolase at any stage of its progress, whereas it only affects dense granule secretion only at its initial stages; these results have been interpreted to show that acid hydrolase secretion requires sustained while dense granule secreion ony requires a brief receptor occupancy (Holmsen et al. JBC 256(1981)9393). The requirement for receptor occupancy in thrombin-induced α-granule secretion and secretion during aggregation have been studied. Increasing concentrations of thrombin were added to gel-fitered platelets containing a constant, high concentration of hirudin. Dense granule secretion was initiated at lower thrombin concentration than those required for α-granule secretion and aggregation; acid hydrolase secretion required higher concentrations. A 14-fold exess of hirudin produced abrupt stop of dense granule secretion and α-granule secretion when added to platelets shortly after thrombin; it had no affect after these secretory process had reached 30% of their maximal values. Acid hydrolase secretion was, however, abruptly stopped by hirudin at any stage. When the platelets were allowed to aggregate, all three secretory processes increased their rates and could now be abruptly stopped by hirudin at any stage. Aggregation (optical) occurred slower than dense granule andoαgranule secretion, and was reversed by hirudin when added before it had reached 30% of its maximum. It is concluded thatαgranule secretion, like dense granule secretion, only requires a short receptor occupancy to be completed, in contrast to the requirement for sustained occupancy for hydrolase secretion.α-granule secretion might, however, require longer occupancy than dense granule secretion. It is possible that aggregation potentiates all secretory responses through close cell contact and that the abrupt inhibition by hirudin of all secretions may have been caused by its effect on the slower aggregation.

Effect Of Prostacyclin (PGI2) On Human Platelet Aggregation And Secretion

1981 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Platelet Rich Plasma ◽  
Dense Granule ◽  
Ionophore A23187 ◽  
Factor V ◽  
Platelet Factor ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Data Representative

PGI2,which increases platelet cAMP(Prostaglandins 13: 389,1977),is a potent inhibitor of aggregation and secretion .We stidued the time course of the same return of platelet function after exposure of platelets to PGI2.Sepharose 2B columns were equilibrated with Tyrode’s albumin buffer, pH7.5 (no Ca2+) containing PGI2 (534nM). Platelet rich plasma was applied and eluted with the same buffer. The filtered platelets(GFP) were then subsampled hourly after elution from the column. Fibrinogen was added to finel concentration of 1.7mg/ml. Platelet aggregation(PA) and release of 14C serotonin (5HT),platelet factor 4(PF4), and factor V (FV) were assayed after stimulation of the platelet by collagen(C), ADP,epinephrine(E), arachidonic acid(AA) and ionophore A23187(I). Data representative of 5 separate studies follow.I(20μg/ml) induced PA was 76%(Ohr),52%(1hr) and 61%(2hr and beyond). Release of 5HT, FV,and PF4 were 60%,1.89u,and 7.97 yg/10 pit, respectively, at time 0 and increased progressively, reaching a plateau at 2 hr. AA(500μg/ml) was 10%(0hr),30%(2hr),68%(3hr) and 8%(4hr). Release of 5HT paralleled PA but release of FV and PF4 remained suppressed for 4 hrs. In contrast α-granule (PF4 and FV)release by C(μg/ml)increased as PA increased while dense granule secretion remained suppressed. PA as well as a and dense granule secretion by ADP (10μM) were minimal during 4 hrs. PA and FV secretion by E (55μM) also remain inhibited for 4 hrs. In spite of this normal dense granule release occurred initially and declined progressively over 4 hours.

scholarly journals Thrombin and ionophore A23187-induced dense granule secretion in storage pool deficient platelets: evidence for impaired nucleotide storage as the primary dense granule defect

Blood ◽  
1983 ◽  
Vol 61 (1) ◽  
pp. 154-162 ◽  
Author(s):  
B Lages ◽  
H Holmsen ◽  
HJ Weiss ◽  
C Dangelmaier
Keyword(s):  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Magnesium Content ◽  
Calcium Pyrophosphate ◽  
Dense Granule ◽  
Ionophore A23187 ◽  
Strongly Correlated ◽  
Storage Pool ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract The secretion of the dense granule constituents ATP, ADP, calcium, pyrophosphate (PPi), and orthophosphate (Pi), and the release of magnesium induced by thrombin and the divalent cation ionophore A23187 have been quantitated directly in gel-filtered platelets from patients with storage pool deficiency (SPD). Both the contents and the maximal amounts of the dense granule constituents secretable by thrombin were decreased in all the patients studied, while the nonsecretable, retained amounts of these substances were identical in SPD and normal platelets. In response to both thrombin and A23187, the amounts of secretable ATP and ADP were strongly correlated in the platelets of individual patients; in contrast, secretable calcium showed no correlation with the nucleotides, and significant amounts of calcium were secreted in the total absence of nucleotide secretion in the platelets of several patients. The contents of magnesium were normal in all patients, and approximately 12% of platelet magnesium was liberated by thrombin in both SPD and normal platelets. A23187 induced the release of up to 70% of the magnesium content of normal platelets, but released significantly less (46%) magnesium from SPD platelets. Platelet aggregation induced by A23187 in platelet-rich plasma was also markedly decreased in SPD platelets. The correlations among secretable dense granule constituents suggest the presence in SPD platelets of abnormal dense granule structures that sequester calcium and other constituents but little or no adenine nucleotides, and are thus consistent with a hypothesis that impaired nucleotide transport and/or storage may be the primary dense granule defect in this disorder. In addition, these results demonstrate that certain responses to A23187 are impaired in SPD platelets.

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