Platelets Are Covercytes, Not Phagocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3533-3533
Author(s):  
James G. White
Keyword(s):  
Tannic Acid ◽  
Platelet Rich Plasma ◽  
Surface Membrane ◽  
Total Surface Area ◽  
Human Platelets ◽  
Osmic Acid ◽  
Platelet Surface ◽  
Flat Surfaces ◽  
Granule Secretion ◽  
Platelet Spreading

Youssefian et al. (Blood2002; 99:4021) have recently reported that engulfment of Staphylococcus Aureus (S.A.) by human platelets is more like phagocytosis by polymorphonuclear leukocytes (PMN) than previously considered.1 Electron microsopy and ultrastructural immunocytochemistry revealed that the engulfment vacuoles containing bacteria were entirely made up of invaginated surface membrane. The surface-connected, open canalicular system (OCS), a tortuous system of interconnected channels continuous with the surface membrane, was not involved in uptake of bacteria. Many of our previous investigations have examined platelet-particle and platelet bacterial interaction, and disagree with the findings of Youssefian et al.1 The present study has used tannic acid as an electron dense tracer to evaluate the hypothesis indicating the OCS is not involved in bacterial uptake.1 Platelets in platelet-rich plasma (PRP) or in washed suspensions (WPS) and samples of PMN were incubated at 37°C with S.A. 502A or S.A. RN 450 for 15, 30, 60, and 120 minutes with or without added ADP.1 Fixation of samples was carried out in glutaraldehyde and osmic acid in the presence of tannic acid. The tannic acid selectively binds to the glycocalyx covering the exterior platelet surface and lining channels of the OCS, and converts osmium to osmium black visible as an electron dense stain. It also combines with fibrinogen/fibrin permitting visualization of α-granule secretion into the OCS. Interaction with bacteria during incubation is variable and infrequent, unless the sample is stirred or an aggregating agent like ADP is added.1 The bacterium attaches itself to the platelet surface, often at or near the opening of an OCS channel. The platelet cannot move the bacterium across the surface as it does small particles up to the size of α granules. Instead the platelet molds itself to the surface, just as it does to large latex spheres and flat surfaces. However, the latter surfaces cannot be internalized, and the platelet continues to spread causing evagination of the OCS to add to the total surface area covered. Bacteria can be internalized. Platelet spreading over the organism causes delilation of the openings of OCS channels. More canaliculi are recruited from the spiderweb of interconnected OCS channels to make a vacuole. Tannic acid staining shows the platelet engulfment vacuoles always contain osmium black around the bacterium, whereas PMN phagocytic vacuoles are never stained. α granules secreting fibrinogen/fibrin into OCS channels stained by tannic acid also discharge their contents into engulfment vacuoles containing bacteria. Direct communication between engulfment vacuoles and the platelet exterior through tannic acid stained OCS canaliculi were identified, while communicating OCS channels were out of the plane of section in other examples. Since the only way tannic acid could stain the engulfment vacuoles containing bacteria was by way of the OCS, it is clear that the OCS is involved in their interiorization. Thus the platelet is a covercyte, not a phagocyte, and its interaction with bacteria is very different from that of the PMN which does not have an OCS.

Differential Ability of Agonists to Express Distinct Pools of Fibrinogen (Gpllb/llla) Receptors which Can Mediate the Aggregation of Human Platelets

1989 ◽  
Vol 62 (03) ◽  
pp. 955-961 ◽  
Author(s):  
Ian S Watts ◽  
Rebecca J Keery ◽  
Philip Lumley
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Surface Membrane ◽  
Blood Platelet ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Platelet Surface ◽  
Human Platelet Aggregation ◽  
Differential Ability ◽  
Blood Platelet Aggregation

SummaryWe have investigated the effect of two procedures that modify human platelet surface membrane glycoprotein (Gp) IIb and IIIa complexes upon whole blood platelet aggregation to a range of agonists. (A) Irreversible disruption of complexes by temporary (30 min) Ca2+-deprivation with EGTA at 37° C. (B) Binding of a monoclonal antibody M148 to the complex. EGTA exposure abolished aggregation to ADP, adrenaline and PAF. In contrast, full aggregation curves to collagen and U-46619 could still be established. EGTA exposure reduced M148 binding to platelets by 80%. Excess M148 abolished aggregation to ADP, PAF, collagen and U-46619. However, upon removal of unbound antibody from platelets full aggregation curves to collagen and U-46619 but not to ADP and PAF could be re-established. Thus human platelet aggregation to ADP, PAF and adrenaline appears absolutely dependent upon surface membrane GpIIb/IIIa complexes. In contrast, collagen and U-46619 cause expression of an additional distinct pool of Gp complexes inaccessible to EGTA and M148 in unstimulated platelets which is intimately involved in aggregation to these agonists.

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.

Activation of human platelets by 2-arachidonoylglycerol is enhanced by serotonin

2003 ◽  
Vol 89 (02) ◽  
pp. 340-347 ◽  
Author(s):  
Monica Bari ◽  
Domenico Del Principe ◽  
Alessandro Finazzi-Agrò ◽  
Mauro Maccarrone
Keyword(s):  
Cyclic Amp ◽  
Cannabinoid Receptors ◽  
Cannabinoid Receptor ◽  
Platelet Rich Plasma ◽  
Maximum Velocity ◽  
Human Platelets ◽  
Platelet Surface ◽  
Therapeutic Implications ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Mechanism

SummaryThe endocannabinoid 2-arachidonoylglycerol (2-AG) has been shown to activate human platelets in platelet-rich plasma, by binding to a “platelet-type” cannabinoid receptor (CBPT). Here, washed human platelets were used to characterize the binding of [3H]2-AG to CBPT, showing a dissociation constant (Kd) of 140 ± 31 nM and a maximum binding (Bmax) of 122 ± 10 pmol.mg protein-1. Selective antagonists of both CB1 and CB2 cannabinoid receptors inhibited this binding, which was enhanced up to ~230% over the controls by 1 µM serotonin (5-hydroxytryptamine, 5-HT). Human platelets were also able to bind [3H]5-HT (Kd = 79 ± 17 nM, Bmax = 14.6 ± 1.3 pmol.mg protein-1), and 1 µM 2-AG enhanced this binding up to ~150%. Moreover, they were able to take up [3H]5-HT through a high affinity transporter (Michaelis-Menten constant = 22 ± 2 nM, maximum velocity = 344 ± 15 pmol.min-1.mg protein-1), which was not affected by 2-AG. Interestingly, 5-HT did not affect the activity of the 2-AG transporter of human platelets. Treatment of washed platelets with 1 µM 2-AG led to increased intracellular inositol-1,4,5-trisphosphate (up to ~300%) and decreased cyclic AMP (down to ~50%). Furthermore, treatment of pre-loaded platelets with 1 µM 2-AG induced a ~300% increase in [3H]2-AG release, according to a CBPT-dependent mechanism. Also, 1 µM 5-HT enhanced the effect of 2-AG on inositol-1,4,5-trisphosphate (~500% of the controls), cyclic AMP (~20%) and [3H]2-AG release (~570%), and the latter process was shown to be partly (~50%) involved in the 5-HT-dependent platelet activation. Taken together, reported findings represent the first demonstration that 2-AG and 5-HT can mutually reinforce their receptor binding on platelet surface, which might have therapeutic implications.

AN EXTRACT OP FEVERFEW INHIBITS INTERACTION OP HUMAN PLATELETS WITH COLLAGEN SUBSTRATES

1987 ◽  
Author(s):  
W Lösche ◽  
A V Mazurov ◽  
W A Groenewegen ◽  
S A Heptinstall ◽  
V S Repin
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Herbal Remedy ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Tanacetum Parthenium ◽  
Ancient Times ◽  
Platelet Spreading ◽  
Dose Dependent ◽  
Scanning Electron

Feverfew (Tanacetum parthenium) has been used since ancient times as a herbal remedy for migraine, fever and arthritis. Recently it has been shown that extracts of feverfew inhibit aggregatory and secretory responses in human platelets induced by various soluble agonists.The interaction of platelets with surfaces coated with human collagens of type III (C III) and IV (C IV) has been studied by measuring the deposition of 51-Cr-labelled platelets and by scanning electron microscopy. Experiments were performed using platelet-rich plasma (PRP) and suspensions of gel-filtered platelets. Platelets were deposited on C III mostly as surface-bound aggregates. In contrast they were deposited on C IV mostly as spread forms of individual cells. Formation of aggregates on C III was more extensive for PRP than for GPP; in contrast platelet spreading on C IV was more extensive for GPP than for PRP.Feverfew extract inhibited the deposition of 51-Cr-labelled platelets on both C III and C IV in a dose dependent way. Similar concentrations of extract were needed to inhibit the formation of surface-bound aggregates and to inhibit platelet spreading in both PRP and GPP.The results indicate that feverfew may have antithrombotic potential in addition to its claimed benefit in certain clinicalconditions.

Platelet Spreading Requires Exocytosis of a Subpopulation of α-Granules Expressing VAMP-7,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3248-3248
Author(s):  
Robert C. Flaumenhaft ◽  
Alan D. Michelson ◽  
Christian G. Peters
Keyword(s):  
Surface Area ◽  
Research Funding ◽  
Data Monitoring Committee ◽  
Time Lapse ◽  
Optical Biosensor ◽  
Total Surface Area ◽  
Dense Granule ◽  
Platelet Surface ◽  
Granule Exocytosis ◽  
Platelet Spreading

Abstract Abstract 3248 There has been recent controversy as to whether platelet α-granules represent a single, homogenous granule population or are composed of different subpopulations that contain different cargos and serve discrete functions. To evaluate this question, we have studied granule movement in spreading platelets. During platelet spreading, the majority of dense and α-granules coalesce in the central granulomere. However, some granules are observed in the platelet periphery. We evaluated whether platelets actively sort a specific subpopulation of α-granules to the periphery during spreading. Granules in spread platelets were stained with antibodies directed at vesicle-associated membrane proteins (VAMPs) −3, −7, and −8. We and others have shown that VAMP-3 and −8 function in granule exocytosis. VAMP-7 contains a profilin-like N-terminal extension capable of interacting with Arp2/3. Samples were imaged using confocal microscopy. Quantitative evaluation of antibody staining in the granulomere vs. the periphery demonstrated that 93+/−7% of VAMP-8, 86+/−14% of von Willebrand factor (an α-granule cargo), and 93+/−7% of serotonin (a dense granule cargo) localized to the central granulomere. In contrast, 77+/−8% of VAMP-7 localized to the periphery. To determine whether a VAMP-7+ α-granule subpopulation actively moves from the granulomere to the platelet periphery during spreading, we loaded platelet α-granules with Qdot 565 nanocrystals and performed time-lapse video microscopy of single platelets. While the majority of endocytosed nanocrystals remained in the central granulomere during spreading, a subpopulation of labeled granules actively moved from the granulomere towards the growing edge at the platelet periphery. Colocalization studies demonstrated that 73% of peripheral Qdot 565 nanocrystals colocalized with VAMP-7. Furthermore, 82% of granules expressing VAMP-7 colocalized with P-selectin (specific for α-granules). These results indicate that a subpopulation of VAMP-7+ α-granules actively moves to the platelet periphery during spreading. To determine whether α-granules are required for spreading, we evaluated spreading in platelets from a patient with gray platelet syndrome (GPS, a congenital absence of α-granules). Platelet surface area increased 4-fold with spreading in normal controls. In contrast, the increase in surface area of GPS platelets following plating for 15 min was minimal – suggesting that α-granules are required for platelet spreading. We also evaluated spreading in murine Jinx platelets, which lack functional Munc13-4. These platelets demonstrate normal morphology and intact proximal signaling, but have a severe granule exocytosis defect. Total surface area of Munc13-4 deficient platelets spread for 15 min was 47+/−3% that of wild-type controls. Platelet spreading was also evaluated using a grated optical biosensor capable of detecting membrane contact with its surface in real time. This technique showed that spreading was decreased in Munc13-4 deficient platelets to 48+/−17% of controls. These results identify a new α-granule subtype that expresses VAMP-7 and is required for platelet spreading. This observation supports the premise that α-granules are heterogeneous and demonstrates that different VAMP isoforms localize to functionally discrete α-granule subpopulations. Disclosures: Michelson: GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Data Monitoring Committee for clinical trial, Research Funding; Takeda: Research Funding.

scholarly journals Effect of anti-P1A1 antibody on human platelets. I. The role of complement

Blood ◽  
1979 ◽  
Vol 53 (4) ◽  
pp. 567-577 ◽  
Author(s):  
DB Cines ◽  
AD Schreiber
Keyword(s):  
Platelet Aggregation ◽  
Complement Activation ◽  
Platelet Rich Plasma ◽  
Plasma Concentrations ◽  
Human Platelets ◽  
Serotonin Release ◽  
Buffer System ◽  
Induce Platelet Aggregation ◽  
Platelet Surface ◽  
Release Reaction

Abstract We studied the interaction of complement with human platelets. Complement was activated by IgG anti-P1A1 antibody obtained from 3 patients with the post-transfusion purpura syndrome. We used a heparin- plasma buffer system that permits complement activation and also preserves platelet function. With this system complement activation was efficient, and platelet immune alteration was extensive. Anti-P1A1 antibody was effective only in the presence of complement, in which case both platelet lysis and serotonin release (release reaction) in the absence of lysis were observed. Platelet lysis, as assessed by 51Cr loss, required 10-fold more antibody than was necessary to induce platelet aggregation and release of 14C-serotonin. This platelet release reaction required an intact classic complement sequence through C6. The extent of platelet serotonin release parallelled the depletion of C1 and C4 from platelet-rich plasma. Concentrations of antibody insufficient to induce platelet aggregation and serotonin release could still activate C1 and deposit increased C3 on the platelet surface. These studies demonstrated that complement activation by anti-P1A1 antibody can alter human platelets in a nonlytic system. Several phases of complement-mediated human platelet alteration are possible, depending on the concentration of anti-P1A1 antibody.

Imer Blocks Phosphorylation of the β3 Integrin, Decreasing Platelet Activation Responses and Protecting Mice From Arterial Thrombosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 189-189
Author(s):  
Brian R. Branchford ◽  
Susan Sather ◽  
Gary Brodsky ◽  
Tara C White-Adams ◽  
Deborah DeRyckere ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Platelet Activation ◽  
Ex Vivo ◽  
Human Platelets ◽  
Fibrillar Collagen ◽  
Carotid Artery Injury ◽  
Platelet Surface ◽  
Β3 Integrin ◽  
Platelet Spreading

Abstract Abstract 189 Background: Growth Arrest Specific gene 6 (Gas6) signaling through platelet-surface Tyro3/Axl/Mer (TAM) receptors leads to platelet activation and thrombus stabilization. This occurs via activation of phosphatidylinositol-3-kinase (PI3K) and Akt, stimulating tyrosine phosphorylation of the β3 integrin. This process amplifies outside-in signaling via αIIbβ3, which is necessary for stable aggregate formation. iMer is a truncated form of the extracellular domain of the Mer receptor tyrosine kinase, produced by alternative splicing, that inhibits Gas6/TAM signaling, likely by acting as a decoy receptor for Gas6. Objectives: We hypothesized that inhibiting the Gas6/TAM pathway with a novel Gas6-sequestering protein would decrease platelet activation responses. We therefore evaluated iMer's inhibition of Gas6 signaling in human and murine platelets in vitro and in vivo. Methods: We measured the inhibitory effect of iMer on platelet activation using laboratory evaluations of platelet function and a murine carotid artery thrombosis model. In vitro studies included aggregometry, adhesion to collagen in a flow chamber, and platelet spreading. These platelet activation responses were tested in human platelets in the presence or absence of the inhibitor and also in wild type (WT) and Gas6/TAM −/− murine platelets. A ferric-chloride model of carotid artery injury was used to compare susceptibility to thrombosis between littermate C57BL/6 mice treated with either iMer or vehicle. Platelet aggregation data was evaluated by the Wilcoxon Signed Rank Test, and times to occlusion following carotid artery injury were compared using the Mann-Whitney Rank Sum Test. Results/Discussion: Western blot analysis demonstrated decreased β3 integrin phosphorylation in iMer-treated human platelets after addition of human Gas6 when compared to controls, consistent with decreased Mer signaling in the presence of iMer. iMer-treated human platelets exhibited significant decreases in ADP- and collagen-induced platelet aggregation. ADP-stimulated samples treated ex vivo with iMer showed an aggregation mean of 74% (SD= +/− 3%), compared to 86% aggregation (+/− 3%) in controls (p=0.016). Collagen-stimulated samples treated ex vivo with iMer exhibited a mean of 70% aggregation (+/− 8%), compared to 88% aggregation (+/−2%) in controls (p=0.004). Electron micrographs of adhered human platelets revealed that iMer delayed, but did not permanently abrogate, platelet spreading on fibrillar collagen (100 μg/mL). Flow cytometric analysis of human platelets showed reduced expression of platelet-surface activation markers (P-selectin and PAC-1) despite stimulation with fibrillar collagen (1 μg/mL). Microfluidic flow assay demonstrated that adhesion of untreated human platelets to collagen at a wall shear rate of 100s−1 resulted in 21.3% (SD=+/− 8%) mean surface area coverage, while ex vivo iMer-treated samples showed only 1.1% (+/− 0.9%) coverage. These results are consistent with those of WT mice compared to that of Gas6/TAM −/−mice in preliminary studies using the same system. Following ferric chloride injury to the carotid artery, 71% of vehicle-treated control mice (n=7) had initial occlusions that remained stable, and only 14% remained patent. In contrast, only 25% of the iMer-treated mice (n=8) formed initial occlusions that remained stable, while 50% remained patent. The iMer treated mice also had a significant decrease (p=0.02) in the duration of first occlusion time (i.e. length of time the initial occlusion lasted), suggesting decreased thrombus stability. Conclusions: iMer is a novel inhibitor of the Gas6/TAM pathway that decreases platelet activation responses and protects mice from arterial thrombosis by decreasing phosphorylation of β3 integrin, which has been shown to be necessary for thrombus stabilization. This compound may, therefore, have translational applications as a novel anti-platelet agent. Disclosures: No relevant conflicts of interest to declare.

Platelet α-Granule Secretion and Cytoskeletal Rearrangements Are Spatially Regulated At the Micro/Nanoscale.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2163-2163
Author(s):  
Yumiko Sakurai ◽  
Yongzhi Qiu ◽  
Byungwook Ahn ◽  
Wilbur Lam
Keyword(s):  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Microcontact Printing ◽  
Morphological Changes ◽  
Human Platelets ◽  
Cell Detection ◽  
Platelet Surface ◽  
Average Area ◽  
Platelet Level ◽  
Granule Secretion

Abstract Abstract 2163 Introduction At sites of vascular injury, activated platelets exhibit dramatic morphological changes and granule secretion to facilitate recruitment of other platelets and clot formation. In our previous work (Kita et al., 2011), we quantified the effect of the microenvironmental geometry on platelet adhesion using microcontact printing and showed that platelet adhesion and spreading is spatially regulated with microscale resolution. Here we demonstrate that platelet secretion of alpha granules, as indicated with P-selectin staining (a marker for a-granules), is also spatially regulated at the micro/nanoscale. Specifically, we show that fibrinogen micropatterns regulate and determine the spatial distribution of P-selectin at the single platelet level. We also show that tubulin, one of the major components of the platelet cytoskeleton, is also rearranged by the nanoscale geometry of the microenvironment. Methods Using microfabrication techniques we previously developed (Kita et al., 2011), patterned polydimethylsiloxane (PDMS) stamps were “inked” with fluorescently-labeled fibrinogen from human plasma. Fibrinogen patterns were then “microstamped” onto glass coverslips and the printed surface was blocked with 1 % BSA. 20 million/ml of washed human platelets were prepared in Tyrode's buffer and incubated onto protein micropatterned surfaces. Immunofluorescence staining was used to visualize P-selectin surface expression and intracellular distribution, and tubulin distribution of adhered platelets. Platelets were also counter-stained with a fluorescent membrane dye or phalloidin for cell detection or cytoskeleton arrangement, respectively. Images were taken via confocal microscopy with a 63x oil immersion objective. Results When washed platelets are incubated onto the micropatterned fibrinogen surface, they generally undergo activation and lamellipodia formation. On uniform, non-patterned fibrinogen glass surfaces, (Fig 1A), the average area of spread platelets is 24.86 μm2(n=30, standard error ± 2.27). When the fibrinogen micropattern was larger than the platelets' average area (Fig 1B), multiple platelets covered the micropattern and followed the microenvironmental geometry with high fidelity. P-selectin was detected on the entire platelet surface at slightly higher concentrations at the margins. However, when the features of the fibrinogen micropattern decreased to 5 μm in diameter (Fig 1C, D, and Fig2), platelet spreading was not constrained within the micropattern boundaries. Interestingly, the area of the platelets that spread beyond the fibrinogen micropattern exhibited much higher P-selectin expression than the areas atop the fibrinogen micropattern (Fig 2, arrows), indicating that expression and distribution of P-selectin are spatially regulated by the geometry of the fibrinogen substrate at the microscale. In addition, when platelets are incubated onto fibrinogen microstamps with densely spaced “holes” (0.4–1.0 μm in diameter) blocked with BSA, platelets fully spread and span over those holes. However, dense P-selectin expression was co-localized with these holes, indicating that P-selectin expression and a-granule release are spatially regulated by the underlying protein micropattern (Fig 3A). Interestingly, tubulin showed the opposite trend and only localized to areas directly above the fibrinogen micropattern (Fig 3B). These observations suggest that both platelet a-granule distribution/secretion and cytoskeleton arrangement are regulated at the single platelet level with nanoscale resolution (Fig 3C). Conclusions Using our platelet adhesion/microstamping technique, we demonstrated that platelets regulate the intracellular trafficking, distribution, and secretion of biomolecules at the nanoscale, responding to the geometry of microenvironment. We will continue to investigate how platelets spatially regulate other aspects of their physiology, including calcium signaling and distribution of receptors/ligands on different substrates such as vWF and collagen. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Further studies of the secretory pathway in thrombin-stimulated human platelets

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1196-1203 ◽  
Author(s):  
JG White ◽  
M Krumwiede
Keyword(s):  
Tannic Acid ◽  
Secretory Pathway ◽  
Surrounding Medium ◽  
Human Platelets ◽  
Secretory Process ◽  
Intense Staining ◽  
Platelet Surface ◽  
Dense Deposits ◽  
Dense Substance ◽  
Secretory Products

Abstract The pathway followed by secretory products stored in platelet alpha granules during the release reaction remains controversial. Tannic acid has been used in the present study as an electron-dense stain to follow the secretory process in thrombin-stimulated platelets. Preliminary experiments demonstrated that tannic acid precipitates fibrinogen, and binds osmium tetroxide to fibrinogen and fibrin strands. Examination of platelets fixed at short intervals after exposure to thrombin and incubated in solutions containing tannic acid revealed electron-dense deposits of osmium not apparent in resting platelets. Granules and lumina of channels making up the open canalicular system (OCS) were unstained in discoid cells. However, exposure to thrombin at concentrations of 1 to 5 U/mL for thirty seconds or more resulted in intense staining of alpha granules by osmium. Some granules communicated directly with dilated channels of the OCS, and several were frequently connected to the same canaliculus. The electron-dense substance in swollen granules and channels appeared to be in the process of extrusion through narrow or dilated openings of the OCS onto the platelet surface. Granule-to-granule fusion and formation of sealed vacuoles of fused granule products unstained by tannic acid-osmium were not observed. The findings support the concept that secretion by stimulated human platelets results from development of direct communications between granules and channels of the OCS and subsequent extrusion of products through channel pores to the surrounding medium.

scholarly journals Further studies of the secretory pathway in thrombin-stimulated human platelets

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1196-1203 ◽  
Author(s):  
JG White ◽  
M Krumwiede
Keyword(s):  
Tannic Acid ◽  
Secretory Pathway ◽  
Surrounding Medium ◽  
Human Platelets ◽  
Secretory Process ◽  
Intense Staining ◽  
Platelet Surface ◽  
Dense Deposits ◽  
Dense Substance ◽  
Secretory Products

The pathway followed by secretory products stored in platelet alpha granules during the release reaction remains controversial. Tannic acid has been used in the present study as an electron-dense stain to follow the secretory process in thrombin-stimulated platelets. Preliminary experiments demonstrated that tannic acid precipitates fibrinogen, and binds osmium tetroxide to fibrinogen and fibrin strands. Examination of platelets fixed at short intervals after exposure to thrombin and incubated in solutions containing tannic acid revealed electron-dense deposits of osmium not apparent in resting platelets. Granules and lumina of channels making up the open canalicular system (OCS) were unstained in discoid cells. However, exposure to thrombin at concentrations of 1 to 5 U/mL for thirty seconds or more resulted in intense staining of alpha granules by osmium. Some granules communicated directly with dilated channels of the OCS, and several were frequently connected to the same canaliculus. The electron-dense substance in swollen granules and channels appeared to be in the process of extrusion through narrow or dilated openings of the OCS onto the platelet surface. Granule-to-granule fusion and formation of sealed vacuoles of fused granule products unstained by tannic acid-osmium were not observed. The findings support the concept that secretion by stimulated human platelets results from development of direct communications between granules and channels of the OCS and subsequent extrusion of products through channel pores to the surrounding medium.

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