scholarly journals Identification of Ca-rich dense granules in human platelets using scanning transmission X-ray microscopy

2019 ◽  
Author(s):  
Tung X. Trinh ◽  
Sook Jin Kwon ◽  
Zayakhuu Gerelkhuu ◽  
Jang-Sik Choi ◽  
Jaewoo Song ◽  
...  
Keyword(s):  
Optical Density ◽  
Human Platelets ◽  
Dense Granule ◽  
Edge Region ◽  
X Ray ◽  
Distribution Maps ◽  
Dense Granules ◽  
Density Maps ◽  
Scanning Transmission ◽  
Contrast Mechanism

ABSTRACTWhole mount (WM) platelet preparations followed by transmission electron microscopy (TEM) observation is the standard method currently used to assess dense granule (DG) deficiency (DGD). However, due to electron density-based contrast mechanism in TEM, other granules such as α-granules might cause false DGs detection. Herein, scanning transmission X-ray microscopy (STXM), was used to identify DGs and minimize false DGs detection of human platelets. STXM image stacks of human platelets were collected at the calcium (Ca) L2,3absorption edge and then converted to optical density maps. Ca distribution maps obtained by subtracting the optical density map at pre-edge region from those obtained at post-edge region were used for identification of DGs based on richness of Ca. Dense granules were successfully detected by using STXM method without false detection based on Ca maps for 4 human platelets. Spectral analysis of granules in human platelets confirmed that DGs contained richer Ca content than other granules. Image analysis of Ca maps provided quantitative parameters which would be useful for developing image-based DG diagnosis models. Therefore, we would like to propose STXM as a promising approach for better DG identification and DGD diagnosis, as a complementary tool to the current WM TEM approach.

scholarly journals Identification of Ca-rich dense granules in human platelets using scanning transmission X-ray microscopy

2020 ◽  
Vol 27 (3) ◽  
pp. 720-724
Author(s):  
Tung X. Trinh ◽  
Sook Jin Kwon ◽  
Zayakhuu Gerelkhuu ◽  
Jang Sik Choi ◽  
Jaewoo Song ◽  
...  
Keyword(s):  
Optical Density ◽  
Human Platelets ◽  
Dense Granule ◽  
Correct Identification ◽  
Edge Region ◽  
X Ray ◽  
Distribution Maps ◽  
Dense Granules ◽  
Density Maps ◽  
Scanning Transmission

Whole-mount (WM) platelet preparation followed by transmission electron microscopy (TEM) observation is the standard method currently used to assess dense granule (DG) deficiency (DGD). However, due to the electron-density-based contrast mechanism in TEM, other granules such as α-granules might cause false DG detection. Here, scanning transmission X-ray microscopy (STXM) was used to identify DGs and minimize false DG detection of human platelets. STXM image stacks of human platelets were collected at the calcium (Ca) L 2,3 absorption edge and then converted to optical density maps. Ca distribution maps, obtained by subtracting the optical density maps at the pre-edge region from those at the post-edge region, were used to identify DGs based on the Ca richness. DGs were successfully detected using this STXM method without false detection, based on Ca maps for four human platelets. Spectral analysis of granules in human platelets confirmed that DGs contain a richer Ca content than other granules. The Ca distribution maps facilitated more effective DG identification than TEM which might falsely detect DGs. Correct identification of DGs would be important to assess the status of platelets and DG-related diseases. Therefore, this STXM method is proposed as a promising approach for better DG identification and diagnosis, as a complementary tool to the current WM TEM approach.

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.

The Appearance Of PF1 And PF3 In Activated Human Platelets

1981 ◽  
Author(s):  
H Sandberg ◽  
A P Bodet ◽  
F A Dombrosei ◽  
L O Andersson ◽  
B R Lentz
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Void Volume ◽  
Factor V ◽  
Dense Granules ◽  
Protein Particles ◽  
Hydrolysis Of ◽  
Platelet Pellet ◽  
Stimulation Of

Collagen and thrombin induced platelet activation were examined, in vitro, with regard to the appearance of surface-associated Factor V-like activity (PF1) and catalytic phospholipid-like surface activity (PF3). Two test systems were used: a clotting assay (a modified KAPTT) and a chromogenic substrate assay (maximum hydrolysis of S-2238). Following stimulation of normal platelets, both PF1 and PF3 appeared simultaneously in the supernatant and platelet pellet. When normal platelets were collected and carefully washed in a buffer containing adenosine, PGE1, and theophylline, the appearance of both PF1 and PF3 was blocked, as was the release of ATP from dense granules, the release of β-TG and PF4 from α-granules, and the occurrence of aggregation. When platelets were collected in this same inhibitor-containing buffer, and then gel filtered/centrifuge-washed in an inhibitor-free buffer, the appearance of PF1 and PF3 was still blocked. This occurred even though release of ATP, β-TG and PF4 as well as aggregation followed a pattern equivalent to platelets never exposed to these inhibitors. When the release supernatant from normal platelets isolated in the absence of inhibitors was gel filtered on Sepharose CL-4B in the presence of EDTA, the carbohydrate-free, lipid- protein particles (70-170nm diam.) that provide PF3 appeared in the void volume. When the release supernatant from normal platelets was gel filtered in the presence of Ca2+, both, PF1 and PF3 eluted in the void volume. With platelets isolated from severe F.V-deficient donors, only PF3 was found in the void volume, in the presence or absence of Ca2+. It seems that the appearance of PF1 and PF3 as coagulant activities is completely separate from both the release of dense granule and α-granule contents as well as platelet aggregation and that the appearance of PF1 requires the presence of Ca2+.

Scanning transmission X-ray microscopy study of subcellular granules in human platelets at the carbon K- and calcium L2,3-edges

Platelets ◽  
2021 ◽  
pp. 1-8
Author(s):  
Jeonghee Shin ◽  
Sehee Park ◽  
Tung X. Trinh ◽  
Sook Jin Kwon ◽  
Jiwon Bae ◽  
...  
Keyword(s):  
Microscopy Study ◽  
Human Platelets ◽  
X Ray ◽  
Scanning Transmission

scholarly journals Diadenosine 5',5'''-p1,p4-tetraphosphate deficiency in blood platelets of the Chediak-Higashi syndrome

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 735-737 ◽  
Author(s):  
BK Kim ◽  
FC Chao ◽  
R Leavitt ◽  
AS Fauci ◽  
KM Meyers ◽  
...  
Keyword(s):  
Whole Blood ◽  
Marked Decrease ◽  
Blood Platelets ◽  
Human Platelets ◽  
Dense Granule ◽  
Atp Level ◽  
Dense Granules ◽  
Moderate Reduction ◽  
Normal Human ◽  
Chediak Higashi Syndrome

Abstract Diadenosine tetraphosphate (AP4A) is an unusual nucleotide found in a variety of cells, including platelets. It has been suggested that platelet AP4A is stored in the dense granules and is metabolically inactive. We have studied the AP4A content of blood platelets in two patients and three cattle with Chediak-Higashi syndrome (CHS), a hereditary platelet defect with dense granule deficiency. Acid-soluble extractions of whole blood and platelets were neutralized. The adenosine triphosphate (ATP) level was measured by luminescence technique. To measure the AP4A content, the neutralized extract was treated with phosphomonoesterase for removal of ATP. The AP4A content was then measured by coupling the phosphodiesterase and luciferase reaction. The AP4A content was 0.43 nmol/mg protein for normal human platelets and 0.004 nmol/mg protein for CHS platelets. The ATP/AP4A ratio was 67 for normal and 3,023 for CHS platelets. The whole blood AP4A was reduced by 89% in CHS patients who had only a slight decrease in ATP level (26% reduction). Similarly, bovine platelets with CHS showed a marked decrease of AP4A content and a moderate reduction of the ATP level. The platelet ATP/AP4A ratio was 351 and 3,133 for normal and CHS cattle, respectively. Results demonstrate a marked reduction of AP4A in CHS platelets and suggest that AP4A may be a useful marker for the measurement of dense granule content in platelets.

Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets

2011 ◽  
Vol 436 (2) ◽  
pp. 469-480 ◽  
Author(s):  
Knut Fälker ◽  
Linda Haglund ◽  
Peter Gunnarsson ◽  
Martina Nylander ◽  
Tomas L. Lindahl ◽  
...  
Keyword(s):  
Receptor Activation ◽  
Human Platelets ◽  
Dense Granule ◽  
Receptor Internalization ◽  
Functional Responses ◽  
Protease Activated Receptors ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Induced Aggregation

PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both Gα12/13 and Gαq signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca2+ mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y12 receptor-induced Gαi signalling accounted for the loss of the aggregation response, as mimicking Gαi/z signalling with 2-MeS-ADP (2-methylthioadenosine-5′-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.

scholarly journals Diadenosine 5',5'''-p1,p4-tetraphosphate deficiency in blood platelets of the Chediak-Higashi syndrome

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 735-737
Author(s):  
BK Kim ◽  
FC Chao ◽  
R Leavitt ◽  
AS Fauci ◽  
KM Meyers ◽  
...  
Keyword(s):  
Whole Blood ◽  
Marked Decrease ◽  
Blood Platelets ◽  
Human Platelets ◽  
Dense Granule ◽  
Atp Level ◽  
Dense Granules ◽  
Moderate Reduction ◽  
Normal Human ◽  
Chediak Higashi Syndrome

Diadenosine tetraphosphate (AP4A) is an unusual nucleotide found in a variety of cells, including platelets. It has been suggested that platelet AP4A is stored in the dense granules and is metabolically inactive. We have studied the AP4A content of blood platelets in two patients and three cattle with Chediak-Higashi syndrome (CHS), a hereditary platelet defect with dense granule deficiency. Acid-soluble extractions of whole blood and platelets were neutralized. The adenosine triphosphate (ATP) level was measured by luminescence technique. To measure the AP4A content, the neutralized extract was treated with phosphomonoesterase for removal of ATP. The AP4A content was then measured by coupling the phosphodiesterase and luciferase reaction. The AP4A content was 0.43 nmol/mg protein for normal human platelets and 0.004 nmol/mg protein for CHS platelets. The ATP/AP4A ratio was 67 for normal and 3,023 for CHS platelets. The whole blood AP4A was reduced by 89% in CHS patients who had only a slight decrease in ATP level (26% reduction). Similarly, bovine platelets with CHS showed a marked decrease of AP4A content and a moderate reduction of the ATP level. The platelet ATP/AP4A ratio was 351 and 3,133 for normal and CHS cattle, respectively. Results demonstrate a marked reduction of AP4A in CHS platelets and suggest that AP4A may be a useful marker for the measurement of dense granule content in platelets.

scholarly journals X-ray ptychographic mode of self-assembled CdSe/CdS octapod-shaped nanocrystals in thick polymers

2020 ◽  
Vol 53 (3) ◽  
pp. 741-747
Author(s):  
Liberato De Caro ◽  
Francesco Scattarella ◽  
Davide Altamura ◽  
Milena P. Arciniegas ◽  
Dritan Siliqi ◽  
...  
Keyword(s):  
Direct Method ◽  
Imaging Method ◽  
Free Standing ◽  
X Ray ◽  
Molecular Weights ◽  
Density Maps ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Processing Techniques ◽  
Pod Length

This work describes the application of X-ray ptychography for the inspection of complex assemblies of highly anisotropic nanocrystals embedded in a thick polymer matrix. More specifically, this case deals with CdSe/CdS octapods, with pod length L = 39 ± 2 nm and pod diameter D = 12 ± 2 nm, dispersed in free-standing thick films (24 ± 4 µm) of polymethyl methacrylate and polystyrene, with different molecular weights. Ptychography is the only imaging method available to date that can be used to study architectures made by these types of nanocrystals in thick polymeric films, as any other alternative direct method, such as scanning/transmission electron microscopy, can be definitively ruled out as a result of the large thickness of the free-standing films. The electron density maps of the investigated samples are reconstructed by combining iterative difference map algorithms and a maximum likelihood optimization algorithm. In addition, post image processing techniques are applied to both reduce noise and provide a better visualization of the material morphological details. Through this process, at a final resolution of 27 nm, the reconstructed maps allow us to visualize the intricate network of octapods inside the polymeric matrices.

scholarly journals Differential energy requirements for platelet responses. A simultaneous study of aggregation, three secretory processes, arachidonate liberation, phosphatidylinositol breakdown and phosphatidate production

1982 ◽  
Vol 208 (1) ◽  
pp. 9-18 ◽  
Author(s):  
Holm Holmsen ◽  
Karen L. Kaplan ◽  
Carol A. Dangelmaier
Keyword(s):  
Energy Charge ◽  
Human Platelets ◽  
Dense Granule ◽  
Metabolic Inhibitors ◽  
Energy Requirements ◽  
Adenylate Energy Charge ◽  
Acid Hydrolases ◽  
Platelet Factor ◽  
Dense Granules ◽  
Collagen Secretion

Previous studies have indicated different energy requirements for some platelet responses; these differences could, however, be due to inadequate methodology and differences in platelet preparation. The present study describes the effect of decreasing ATP availability on seven platelet responses measured in gel-filtered human platelets. The cells, prelabelled with 5-hydroxy[3H]tryptamine, [3H]- or [14C]adenine, [32P]Pi or [3H]arachidonate, were incubated with antimycin A and 2-deoxy-d-glucose. Platelet responses induced by thrombin and collagen (secretion only), level of metabolic ATP and the adenylate energy charge (AEC) were determined at various times during incubation. Platelet aggregation was rapidly inhibited after a lag of 5–15 min and with 50% inhibition at AEC = 0.55–0.60. Secretion of 5-hydroxy[14C]tryptamine and ATP + ADP from dense granules and of fibrinogen and β-thromboglobin from α-granules were inhibited in parallel, without a lag and with 50% inhibition at AEC = 0.65–0.70. The inhibition of secretion of platelet factor 4 from the α-granules followed another pattern with 50% inhibition at AEC = 0.70–0.80. Breakdown of [3H]-phosphatidylinositol, formation of [3H]- and [32P]-phosphatidate, liberation of [3H]arachidonate and secretion of acid hydrolases were inhibited in parallel and inhibition was present at the start of incubation with 50% inhibition at AEC = 0.80–0.87. These results suggest that the responses have different energy requirements, increasing in the order: aggregation < dense granule and α-granule secretion < acid hydrolase secretion, phosphatidylinositol breakdown, phosphatidate formation and arachidonate liberation. The powerful inhibition of phosphatidylinositol breakdown by metabolic inhibitors suggests that energy-requiring steps are involved in the activation of phospholipase C.

Secreted Dense Granule Adenine Nucleotides Promote Calcium Influx and the Maintenance of Elevated Cytosolic Calcium Levels in Stimulated Human Platelets

1999 ◽  
Vol 81 (02) ◽  
pp. 286-292 ◽  
Author(s):  
Harvey Weiss ◽  
Bruce Lages
Keyword(s):  
Calcium Influx ◽  
Adenine Nucleotides ◽  
Normal Subjects ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Dense Granule ◽  
Specific Component ◽  
Fura 2 ◽  
Dense Granules ◽  
Platelet Storage

SummaryEvidence that secreted dense granule adenine nucleotides mediate part of the agonist-induced cytosolic calcium ([Ca2+]i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca2+]i in creases induced by thrombin and the endoperoxide analog U46619, but a significantly enhanced decay of elevated [Ca2+]i levels following the initial increases. With thrombin, this enhanced [Ca2+]i decay was associated with decreased Ca2+ influx, as measured by Mn2+ quench of fura-2 fluorescence. Addition of micromolar concentrations of ADP, alone or together with ATP, after stimulation reversed the enhanced [Ca2+]I decay and increased Mn2+ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of ATP or apyrase before stimulation increased [Ca2+]I decay and decreased Mn2+ quench in normal platelets, but had little effect in SPD platelets. ATP and α,β-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn2+ quench, but to lesser extents than did ADP, in SPD platelets isolated and loaded with fura-2 in the presence of apyrase. Similar effects of ADP and excess ATP were seen in U46619-stimulated platelets, but decreased Ca2+ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with U46619. These results suggest that secreted dense granule ADP and ATP contribute to the maintenance of elevated [Ca2+]i levels, but not to the initial [Ca2+]i increases, in stimulated human platelets, most likely via a nucleotide-specific component of Ca2+ influx which may be mediated by interactions with both P2X1 and P2Y1 purinoceptors.

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