A MORPHOLOGICAL APPROACH TO THE MECHANISMS OF RISTOCETIN INDUCED PLATELET AGGLUTINATION(RIPA)

1987 ◽  
Author(s):  
G Escolar ◽  
J Monteagudo ◽  
N Villamor ◽  
M Garrido ◽  
R Castillo
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Complex Interaction ◽  
Electron Dense Deposit ◽  
Transmission Electron ◽  
Dense Deposit ◽  
Morphological Approach ◽  
Adhesive Proteins

Changes in the morphology of human platelets induced by Ristocetin (RIPA) have been analyzed at ultraestructural level by means of a tannine acid procedure. Studies were completed with aggregation and binding experiments. Modificationsof these tests induced by apyrase,a monoclonal antibody (Mab) to GPIIb/IIIa and EDTA were also investigated.Transmission electron microscopy reveals that ristocetin precipitates adhesive proteins on plateletmembrane. An electron-dense deposit was noticeable within 20 secondsafter ristocetin was added. When experiments were carried out in theaggregometer cuvette under stirring, groups of platelets become activated, change shape, and finally aggregate releasing part of their content. The morphology of aggregates did not differ from those formed in the presence of ADP.Aggregation studies demonstrated that a Mab to GPIIb/IIIa modifies the extent and the rate of the aggregation curve when RIPA was performed in citrated platelet rich plasma (c-PRP). Apyrase modified the extent but not the slope of the curve. Neither the antibody nor apyrase modified RIPA when it was performed in PRP obtained in presence of EDTA. Binding experiments confirmed that I-vWF bound to platelets in presence of ristocetin was not modified by apyrase or anti-GPIIb/IIIa Mab. All these facts together suggest that RIPA,when performed in c-PRP besides reflecting the interaction of GPI with vWF, is also testing other mechanisms of the platelet function including exposure of GPIIb/IIIa complex, interaction of fibrinogen with this glycoprotein, and the contribution of the release reaction.

scholarly journals A novel cytochemical marker for liposome decomposition in lysosomes.

1983 ◽  
Vol 31 (3) ◽  
pp. 404-410 ◽  
Author(s):  
S C Ho ◽  
L Huang
Keyword(s):  
Electron Microscopy ◽  
Enzymatic Reaction ◽  
Chinese Hamster ◽  
Electron Dense Deposit ◽  
Transmission Electron ◽  
Dense Deposit ◽  
Condensed Product ◽  
Dense Deposits ◽  
Indigo Blue ◽  
Cytochemical Marker

The endocytosis of large unilamellar liposomes composed of phosphatidylcholine by the cultured Chinese hamster V-79 cells is demonstrated with electron microscopy cytochemistry. A novel cytochemical marker, 5-Br,4-Cl,3-indolylphosphate (BCIP) is used. This marker is a soluble and colorless substrate for the lysosomal acid phosphatase and can be readily entrapped in liposomes. The product of the enzymatic reaction, 5-Br,4-Cl,3-hydroxy indole, rapidly self-condenses and becomes an insoluble derivative of indigo blue. In thin section transmission electron microscopy, the condensed product appears as electron-dense deposits in the lysosomes. Since the electron-dense deposit only appears when the endocytosed liposomes are delivered to the lysosomes as the result of phagosome-lysosome fusion, this marker provides a unique cytochemical means to reveal those liposomes that are lysosomotropic and are actually decomposed within the lysosomes. No electron-dense deposits are found in the liposome-treated cells in the presence of chloroquine, or a combination of NaN3 and deoxyglucose. As a comparison, we have also used horseradish peroxidase entrapped in liposomes to confirm the endocytic uptake of liposomes. Using a radioactive marker, 125I-labeled lysozyme, entrapped in liposomes, it is shown that about 20-30% of liposome uptake by V-79 cells is due to endocytosis.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Adhesion of Phytophthora palmivora zoospores: electron microscopy of cell attachment and cyst wall fibril formation

1975 ◽  
Vol 18 (1) ◽  
pp. 123-132
Author(s):  
V.O. Sing ◽  
S. Bartnicki-Garcia
Keyword(s):  
Electron Microscopy ◽  
Cell Adhesion ◽  
Cyst Wall ◽  
Cell Attachment ◽  
Sodium Dodecyl ◽  
Film Surface ◽  
Phytophthora Palmivora ◽  
Thin Sections ◽  
Electron Dense Deposit ◽  
Dense Deposit

Zoospores of Phytophthora palmivora adhered to a plastic film surface were examined by electron microscopy. Three stages of adhesion were compared: (1) non-adhesive, unencysted zoospores, (2) adhered incipient cysts, and (3) adhered mature cysts. Thin sections of incipient cysts revealed cells attached to the film surface through the partially discharged contents of the so-called peripheral vesicles; this seems to be the first step in cell adhesion. In mature cysts, the adhesive appeared to have been compacted into an electron-dense deposit binding the cyst wall to the plastic surface. The adhesion zone was also examined in face view after lysing attached incipient cysts with sodium dodecyl sulphate. Cyst wall microfibrils were seen together with an amorphous substance (presumably the adhesive material). The microfibrils were in various stages of formation. Seemingly, adhesion and microfibril formation take place concurrently. The possibility was considered that the material contained in the peripheral vesicles serves in both cell adhesion and microfibril elaboration.

The death of human platelets during incubation in citrated plasma involves shedding of CD42b and aggregation of dead platelets

2006 ◽  
Vol 95 (01) ◽  
pp. 100-106 ◽  
Author(s):  
John Savill ◽  
Simon Brown ◽  
Paul Hartley
Keyword(s):  
Platelet Rich Plasma ◽  
Vital Staining ◽  
Human Platelets ◽  
Staining Procedure ◽  
Aggregate Formation ◽  
Factors Affecting ◽  
Transmission Electron ◽  
Reliable Assessment ◽  
Vital Stains ◽  
Calcein Am

SummaryThe ability to readily identify dead platelets is invaluable to studies examining the means of their death, factors affecting their lifespan and their means of clearance by phagocytes. The aim of the present work was to develop a vital staining procedure for the rapid and objective discrimination of live from dead platelets that accrued in citrated platelet rich plasma (cPRP) incubated at 37°C for several days. By transmission electron microscopy it was noted that platelet death was morphologically similar to necrosis and associated with aggregate formation. The vital dyes calcein-AM and FM 4–64 were found to robustly report the death of platelets and indicated that the aggregates which formed during incubation were populated exclusively by dead platelets. Additionally, platelet death was associated with the shedding of CD42b. Microscopic and cytometric analyses of incubated cPRP indicated that shedding of CD42b and aggregate formation by dead platelets were completely inhibited by the metalloproteinase inhibitor GM6001. Automated counting of platelets incubated in the presence of GM6001 revealed that death did not lead to a loss in cellularity. It is proposed that calcein-AM and FM4–64 are effective as vital stains for the reliable assessment of platelet viability and that platelet aggregation can occur by a novel mechanism dependent upon platelet death and metalloproteinase activity.

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.

scholarly journals Platelet-melanoma cell interaction is mediated by the glycoprotein IIb- IIIa complex

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 658-663
Author(s):  
H Boukerche ◽  
O Berthier-Vergnes ◽  
E Tabone ◽  
JF Dore ◽  
LL Leung ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Melanoma Cell ◽  
Melanoma Cell Line ◽  
Cell Interaction ◽  
Melanoma Cells ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Human Malignant Melanoma

A human malignant melanoma cell line (M3Dau) was observed by electron microscopy to interact directly with human platelets and induced platelet aggregation. Fab fragments of a monoclonal antibody MoAb (LYP18), directed against the platelet glycoprotein (GP) IIb-IIIa complex, inhibited platelet-melanoma interactions and platelet-platelet aggregation. M3Dau melanoma cells bind LYP 18 and synthesize IIb-IIIa- like GPs. When the melanoma cells were preincubated with LYP 18, tumor- platelet interaction did not occur, suggesting that the interaction may be mediated by the IIb-IIIa-like GPs present on the melanoma cell surface. Glanzmann's thrombasthenic platelets, lacking GPIIb and IIIa, did not interact with melanoma cells, indicating that the platelet GPIIb-IIIa complex is also necessary for the platelet-melanoma cell interaction. This work demonstrates the importance of the IIb-IIIa-like GPs, present on M3Dau melanoma cells, in mediating tumor-platelet interactions.

scholarly journals mAb CZP-315.D9: An Antirecombinant Cruzipain Monoclonal Antibody That Specifically Labels the Reservosomes ofTrypanosoma cruziEpimastigotes

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Cassiano Martin Batista ◽  
Lia Carolina Soares Medeiros ◽  
Iriane Eger ◽  
Maurilio José Soares
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Cysteine Proteinase ◽  
Igg Antibody ◽  
Western Blots ◽  
Myeloma Cells ◽  
Transmission Electron ◽  
Large Round ◽  
Endocytosis Pathway ◽  
Limiting Dilution

Reservosomes are large round vesicles located at the posterior end of epimastigote forms of the protozoanTrypanosoma cruzi, the etiological agent of Chagas disease. They are the specific end organelles of the endocytosis pathway ofT. cruzi, and they play key roles in nutrient uptake and cell differentiation. These lysosome-like organelles accumulate ingested macromolecules and contain large amounts of a major cysteine proteinase (cruzipain or GP57/51 protein). Aim of this study was to produce a monoclonal antibody (mAb) against a recombinantT. cruzicruzipain (TcCruzipain) that specifically labels the reservosomes. BALB/c mice were immunized with purified recombinant TcCruzipain to obtain the mAb. After fusion of isolated splenocytes with myeloma cells and screening, a mAb was obtained by limiting dilution and characterized by capture ELISA. We report here the production of a kappa-positive monoclonal IgG antibody (mAb CZP-315.D9) that recognizes recombinant TcCruzipain. This mAb binds preferentially to a protein with a molecular weight of about 50 kDa on western blots and specifically labels reservosomes by immunofluorescence and transmission electron microscopy. The monoclonal CZP-315.D9 constitutes a potentially powerful marker for use in studies on the function of reservosomes ofT. cruzi.

The Prevalence of Mesangial Electron-Dense Deposits in PLA2R-Positive Membranous Nephropathy

Nephron ◽  
2021 ◽  
pp. 1-5
Author(s):  
Gabriel Giannini ◽  
Lois J. Arend
Keyword(s):  
Electron Microscopy ◽  
Nephrotic Syndrome ◽  
Membranous Nephropathy ◽  
Immunofluorescence Microscopy ◽  
Common Cause ◽  
Electron Dense Deposit ◽  
Dense Deposit ◽  
Phospholipase A2 Receptor ◽  
Dense Deposits ◽  
Negative Biopsies

<b><i>Introduction:</i></b> Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults and can be primary or secondary. The antigenic target of antibodies in 70% of primary cases is phospholipase A2 receptor (PLA2R). The presence or absence of mesangial electron-dense deposits has been used to distinguish between primary and secondary MN. Mesangial deposits suggest MN due to lupus, infection, or other causes, though they are reported to occur in approximately 10% of primary MN. Staining for PLA2R is now frequently used for confirming a diagnosis of primary MN. If mesangial deposits predict a secondary cause, they should be more frequent in PLA2R-negative biopsies. <b><i>Methods:</i></b> A review of institutional kidney biopsies between March 2017 and June 2020 identified all cases of MN. Cases with a diagnosis of lupus or near “full-house” staining by immunofluorescence microscopy (IF) were excluded. Light microscopy, IF, and electron microscopy (EM) were performed. PLA2R staining was performed by IF. EM for all cases was reviewed and electron-dense deposit location, distribution, and size were determined. <b><i>Results:</i></b> Ninety-three cases of MN were identified, of which 86 had both PLA2R staining and EM performed. Of these, 51 cases (59%) were positive for PLA2R and 35 (41%) were negative. Mesangial electron-dense deposits were present in 22 (25.6%) of the 86 cases, including 27.5% (14/51) of PLA2R-positive cases and 22.8% (8/35) of PLA2R-negative cases. No difference was seen in size or distribution of deposits, or other features considered suggestive of secondary MN. <b><i>Conclusion:</i></b> PLA2R-negative cases were not more likely to have mesangial deposits than PLA2R-positive cases. Mesangial deposits should not be used as an indicator of secondary MN.

Microstructure and misfit relaxation in SrTiO3/SrRuO3 bilayer films on LaAlO3(100) substrates

2001 ◽  
Vol 16 (12) ◽  
pp. 3443-3450 ◽  
Author(s):  
J. S. Wu ◽  
C. L. Jia ◽  
K. Urban ◽  
J. H. Hao ◽  
X. X. Xi
Keyword(s):  
Electron Microscopy ◽  
Elastic Strain ◽  
Stacking Faults ◽  
Structure Change ◽  
Complex Interaction ◽  
Planar Defects ◽  
Bilayer Films ◽  
Partial Dislocations ◽  
Transmission Electron ◽  
Misfit Accommodation

We studied the microstructure of SrTiO3/SrRuO3 bilayer films on (001) LaAlO3 substrates by high-resolution transmission electron microscopy. At the SrRuO3/LaAlO3 interface a defect configuration of stacking faults and nanotwins bounding either Frank partial dislocations or Shockley partial dislocations and complex interaction between these planar defects were found to be the dominant means of misfit accommodation. The misfit in the SrTiO3/SrRuO3 system, however, is mainly accommodated by elastic strain. Most of the observed defects in the SrTiO3 layer can be related to the [111] planar defects in the SrRuO3 layer propagating and reaching the SrTiO3/SrRuO3 interface. Furthermore, a [110] planar defect can also be introduced in the SrTiO3 layer due to the structure change of the SrTiO3/SrRuO3 interface.

scholarly journals Platelet-melanoma cell interaction is mediated by the glycoprotein IIb- IIIa complex

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 658-663 ◽  
Author(s):  
H Boukerche ◽  
O Berthier-Vergnes ◽  
E Tabone ◽  
JF Dore ◽  
LL Leung ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Melanoma Cell ◽  
Melanoma Cell Line ◽  
Cell Interaction ◽  
Melanoma Cells ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Human Malignant Melanoma

Abstract A human malignant melanoma cell line (M3Dau) was observed by electron microscopy to interact directly with human platelets and induced platelet aggregation. Fab fragments of a monoclonal antibody MoAb (LYP18), directed against the platelet glycoprotein (GP) IIb-IIIa complex, inhibited platelet-melanoma interactions and platelet-platelet aggregation. M3Dau melanoma cells bind LYP 18 and synthesize IIb-IIIa- like GPs. When the melanoma cells were preincubated with LYP 18, tumor- platelet interaction did not occur, suggesting that the interaction may be mediated by the IIb-IIIa-like GPs present on the melanoma cell surface. Glanzmann's thrombasthenic platelets, lacking GPIIb and IIIa, did not interact with melanoma cells, indicating that the platelet GPIIb-IIIa complex is also necessary for the platelet-melanoma cell interaction. This work demonstrates the importance of the IIb-IIIa-like GPs, present on M3Dau melanoma cells, in mediating tumor-platelet interactions.

Sign in / Sign up

Export Citation Format

Share Document