Histochemical and Ultrastructural study of the Platelet-Arterial Subendothelium Interaction

1979 ◽  
Author(s):  
Y.J. Legrand ◽  
J. Bariety ◽  
P. Birembaut ◽  
H. Michel ◽  
F. Fauvel ◽  
...  
Keyword(s):  
Tannic Acid ◽  
Platelet Adhesion ◽  
Ricinus Communis ◽  
Blood Perfusion ◽  
Rabbit Aorta ◽  
Human Platelets ◽  
Ruthenium Red ◽  
Ultrastructural Studies ◽  
Bacterial Collagenase ◽  
Before And After

In order to characterize the subendothelial macromolecules able to interact with platelets rabbit aortas were de-endothelialized and incubated either with chymotrypsin (24 h at 37° C), with a highly purified bacterial collagenase (2 h at 37° C), or with chymotrypsin followed by collagenase or the reverse. Histochemical ultrastructural studies were performed before and after blood perfusion, using three different staining procedures: tannic acid (for the microfibrillar structures and elastin), ruthenium red (for the polyanions) and two peroxidase-labelled lectins (concanavalin A and ricinus communis (for the detection of saccharidic determinants)). After chymotrypsin, the tannic acid +ve, ruthenium red +ve and lectin +ve microfibrils had been digested; platelet adherence to the remaining sparse collagen fibrils persisted. After collagenase, these microfibrils are preserved, collagen has been digested, but the platelets can still adhere. After combined incubation with both enzymes, elastin is the only remaining constituent and no platelet adhesion can be observed.These results show that in the rabbit aorta subendothelium besides collagen, microfibrillar glycoproteins are able to interact with human platelets.

Histochemical And Ultrastructural Study Of The Platelet-Arterial Subendothelium Interaction

1979 ◽  
Author(s):  
Y Legrand ◽  
J Bariety ◽  
P Birembaut ◽  
H Michel ◽  
F Fauvel ◽  
...  
Keyword(s):  
Tannic Acid ◽  
Platelet Adhesion ◽  
Ricinus Communis ◽  
Blood Perfusion ◽  
Rabbit Aorta ◽  
Human Platelets ◽  
Ruthenium Red ◽  
Ultrastructural Studies ◽  
Bacterial Collagenase ◽  
Before And After

In order to characterize the subendothelial macromolecules able to interact with platelets rabbit aortas were de-endothelialized and incubated either with chymotrypsin (24 h at 37° C), with a highly purified bacterial collagenase (2 h at 37° C), or with chymotrypsin followed by collagenase or the reverse. Histochemical ultrastructural studies were performed before and after blood perfusion, using three different staining procedures : tannic acid (for the microfibrillar structures and elastin), ruthenium red (for the polyanions) and two peroxidase-labelled lectins (concanavalin A and ricinus communis (for the detection of saccharidic determinants)). After chymotrypsin, the tannic acid +ve, ruthenium red +ve and lectin +ve microfibrils had been digested ; platelet adherence to the remaining sparse collagen fibrils persisted. After collagenase, these microfibrils are preserved, collagen has been digested, but the platelets can still adhere. After combined incubation with both enzymes, elastin is the only remaining constituent and no platelet adhesion can be observed.These results show that in the rabbit aorta subendothelium besides collagen, microfibrillar glycoproteins are able to interact with human platelets.

THE BEHAVIOUR OF RABBIT PLASMINOGEN AT THE LUMINAL SURFACE OF RABBIT AORTA IN VIVO BEFORE AND AFTER BALLOON-CATHETER INJURY

1987 ◽  
Author(s):  
Mark W C Hatton ◽  
Susan Moar ◽  
Mary Richardson
Keyword(s):  
White Male ◽  
Balloon Catheter ◽  
Rabbit Aorta ◽  
Ruthenium Red ◽  
Transmission Electron ◽  
Before And After ◽  
Ruthenium Red Staining ◽  
Balloon Catheter Injury

A previous study from this laboratory has identified the susceptibility of the de-endothelialised aorta, particularly the proteoglycan (PG) components of the extracellular matrix (ECM), to proteolytic damage if exposed to plasmin in vitro. To explore the possiblity that this occurs in vivo, a possible association between 125I-plasminogen (PLG) binding to the arterial wall, its activation to plasmin (PLN) and, subsequently, proteolytic damage to the intimal ECM has been studied. Intravenous injection of 125I-PLG in healthy N.Z. white male rabbits showed that PLG associated minimally (<0.01% of circulating PLG/cm2 /ml blood at 1 h) with the thoracic aorta endothelium, measured after Hautchen preparation from 1-cm vessel segments. Trans endothelial passage, measured as 125I-PLG associated with thg subendothelium (intima-media), progressed to 0.015%/cm2 /ml blood at 1 h. In contrast, the process of de-endothelialisation by balloon catheter led to a rapid uptake of bI-PLG by the denuded vessel surface. At saturation (approx. 10 min after injury), 0.7 - 0.8% of circulating PLG/cm2/ml blood was adsorbed by the entire de-endothelialised intima-media: Of the adsorbed PLG, 2-3% was associated with the platelet layer. Uptake was not inhibited by eACA (dose: 200 mg/kg) given i.v. before I-PLG. Adsorbed PLG was not released significantly from segments incubated in MEM containing 4% (w/v) RSA in vitro PLN activity was not detected. Furthermore, assessment of the ECM by transmission electron microscopy, after ruthenium red staining, showed that uptake of PLG by the de-endothelialised vessel in vivo was not associated with obvious damage to the PG components. Supported by the Heart and Stroke Foundation of Ontario.

scholarly journals Histochemical and ultrastructural characterization of subendothelial glycoprotein microfibrils interacting with platelets.

1982 ◽  
Vol 30 (1) ◽  
pp. 75-80 ◽  
Author(s):  
P Birembaut ◽  
Y J Legrand ◽  
J Bariety ◽  
R Bretton ◽  
F Fauvel ◽  
...  
Keyword(s):  
Concanavalin A ◽  
Ricinus Communis ◽  
Blood Platelets ◽  
Ruthenium Red ◽  
Fibrillar Collagen ◽  
Bacterial Collagenase ◽  
Ultrastructural Characterization ◽  
Human Blood Platelets

The interaction of human blood platelets with collagenase-treated rabbit subendothelium was studied by histochemical ultrastructural methods and by morphometric semi-quantitative analysis. Aortas were deendothelialized and incubated: 1) with a highly purified bacterial collagenase whose specificity was controlled; and 2) with the same collagenase followed by chymotrypsin. For histochemical studies, tannic acid, ruthenium red, and peroxidase-labeled Ricinus communis and concanavalin A were used. Electron microscopy showed that after digestion of fibrillar collagen by collagenase, adherent and aggregated platelets were observed on Ricinus communis-, concanavalin A-, and ruthenium red-positive glycoprotein microfibrils. After successive incubation with collagenase and chymotrypsin, the microfibrils disappeared. No platelets were observed on the remnant amorphous elastin. Morphometric analysis confirmed the interaction of platelets with collagenase-treated subendothelium. In addition, glycoproteins were extracted from collagenase-treated rabbit aortas using 5 M guanidine. Using an in vitro quantitative test, significant platelet adhesion to these glycoproteins was observed. Our results show an interaction between platelets and noncollagenic glycoprotein microfibrils.

Effect of Aspirin and Dipyridamole on the Interaction of Human Platelets with Sub-Endothelium: Studies Using Citrated and Native Blood

1981 ◽  
Vol 45 (02) ◽  
pp. 136-141 ◽  
Author(s):  
Harvey J Weiss ◽  
Vincent T Turitto ◽  
William J Vicic ◽  
Hans R Baumgartner
Keyword(s):  
Shear Rate ◽  
Platelet Adhesion ◽  
Inner Core ◽  
Thrombus Formation ◽  
Human Subjects ◽  
Rabbit Aorta ◽  
Blood Platelet ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Platelet Thrombi

SummaryThe effect of aspirin and dipyridamole ingestion on the interaction of platelets with the subendothelium was studied using both citrated blood and directly sampled (native) blood. After obtained control studies, normal human subjects ingested 0.6 g of aspirin, 150 mg of dipyridamole, or a placebo and studies were repeated 1½ hrs later. Subjects continued on placebo, aspirin (0.6 g b.i.d.) or dipyridamole (100 mg q.i.d.) for 6 days and studies were obtained 1½ hrs after the last dose. Blood was circulated through an annular chamber on whose inner core were mounted everted segments of de-endothelialized rabbit aorta. The wall shear rate was 2,600 sec-1. Surface coverage with adherent platelets and platelet thrombi, as well as several parameters of thrombus dimensions, were evaluated morphometrically. Aspirin ingestion markedly reduced platelet thrombi in citrated blood, – but had a much lesser inhibitory effect in native blood. Platelet adhesion was unaffected in native blood; it was slightly decreased in citrated blood, in contrast to previous findings in which a lower shear rate (800 sec-1) was used. Ingestion of dipyridamole did not inhibit platelet adhesion or thrombi in either citrated or native blood. The studies indicate that, with the flow conditions used, aspirin is a relatively weak inhibitor of platelet thrombus formation in directly sampled human blood.

The Cellular Origin in Atheromatous Lesion

1970 ◽  
Vol 28 ◽  
pp. 202-203
Author(s):  
T. M. Murad ◽  
H. A. I. Newman ◽  
K. F. Kern
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Rabbit Aorta ◽  
Mixed Population ◽  
The Other ◽  
Cellular Origin ◽  
Ultrastructural Studies ◽  
Atherosclerotic Lesions ◽  
Show Evidence ◽  
Early Lesion

The origin of lipid containing cells in atheromatous lesion has been disputed. Geer in his study on atheromatous lesions of rabbit aorta, suggested that the early lesion is composed mainly of lipid-laden macrophages and the later lesion has a mixed population of macrophages and smooth muscle cells. Parker on the other hand, was able to show evidence that the rabbit lesion is primarily composed of lipid-laden cells of smooth muscle origin. The above studies and many others were done on an intact lesion without any attempt of cellular isolation previous to their ultrastructural studies. Cell isolation procedures have been established for atherosclerotic lesions through collagenase and elastase digestion Therefore this procedure can be utilized to identify the cells involved in rabbit atheroma.

The Fine Structure of the Sheath of Volvox Carteri f. Weismannia

1977 ◽  
Vol 35 ◽  
pp. 346-347
Author(s):  
Regina Birchem
Keyword(s):  
Developmental Stages ◽  
Ruthenium Red ◽  
Sulfated Polysaccharides ◽  
Volvox Carteri ◽  
High Voltage Electron Microscopy ◽  
Ultrastructural Studies ◽  
Voltage Electron ◽  
Sheath Formation ◽  
And Inversion ◽  
Sheath Material

Spheroids of the green colonial alga Volvox consist of biflagellate Chlamydomonad-like cells embedded in a transparent sheath. The sheath, important as a substance through which metabolic materials, light, and the sexual inducer must pass to and from the cells, has been shown to have an ordered structure (1,2). It is composed of both protein and carbohydrate (3); studies of V. rousseletii indicate an outside layer of sulfated polysaccharides (4).Ultrastructural studies of the sheath material in developmental stages of V. carteri f. weismannia were undertaken employing variations in the standard fixation procedure, ruthenium red, diaminobenzidine, and high voltage electron microscopy. Sheath formation begins after the completion of cell division and inversion of the daughter spheroids. Golgi, rough ER, and plasma membrane are actively involved in phases of sheath synthesis (Fig. 1). Six layers of ultrastructurally differentiated sheath material have been identified.

Radiolabeled r-Hirudin as a Measure of Thrombin Activity at, or within, the Rabbit Aorta Wall In Vitro and In Vivo

1994 ◽  
Vol 71 (04) ◽  
pp. 499-506 ◽  
Author(s):  
Mark W C Hatton ◽  
Bonnie Ross-Ouellet
Keyword(s):  
Rabbit Aorta ◽  
Balloon Injury ◽  
Recombinant Hirudin ◽  
Aorta Wall ◽  
Thrombin Activity ◽  
Before And After ◽  
The Matrix

SummaryThe behavior of 125I-labeled recombinant hirudin towards the uninjured and de-endothelialized rabbit aorta wall has been studied in vitro and in vivo to determine its usefulness as an indicator of thrombin activity associated with the aorta wall. Thrombin adsorbed to either sulfopropyl-Sephadex or heparin-Sepharose bound >95% of 125I-r-hirudin and the complex remained bound to the matrix. Binding of 125I-r-hirudin to the exposed aorta subendothelium (intima-media) in vitro was increased substantially if the tissue was pre-treated with thrombin; the quantity of l25I-r-hirudin bound to the de-endothelialized intima-media (i.e. balloon-injured in vitro) correlated positively with the quantity of bound 131I-thrombin (p <0.01). Aortas balloon-injured in vivo were measured for thrombin release from, and binding of 125I-r-hirudin to, the de-endothelialized intimal surface in vitro; 125I-r-hirudin binding correlated with the amount of active thrombin released (p <0.001). Uptake of 125I-r-hirudin by the aorta wall in vivo was proportional to the uptake of 131I-fibrinogen (as an indicator of thrombin activity) before and after balloon injury. After 30 min in the circulation, specific 125I-r-hirudin binding to the uninjured and de-endo- thelialized (at 1.5 h after injury) aorta wall was equivalent to 3.4 (± 2.5) and 25.6 (±18.1) fmol of thrombin/cm2 of intima-media, respectively. Possibly, only hirudin-accessible, glycosaminoglycan-bound thrombin is measured in this way.

Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

1994 ◽  
Vol 71 (01) ◽  
pp. 091-094 ◽  
Author(s):  
M Cattaneo ◽  
B Akkawat ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
C Cimminiello ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Before And After ◽  
Normal Human ◽  
Platelet Aggregates ◽  
Induced Aggregation

SummaryNormal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80–90% 14C-serotinin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5× the activity of thrombin) and PGE1 (10 μmol/1) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 μmol/1) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

Further Evidence that Glycoprotein IIb-IIIa Mediates Platelet Spreading on Subendothelium

1991 ◽  
Vol 65 (02) ◽  
pp. 202-205 ◽  
Author(s):  
Harvey J Weiss ◽  
Vincet T Turitto ◽  
Hans R Baumgartner
Keyword(s):  
Platelet Adhesion ◽  
Surface Coverage ◽  
Rabbit Aorta ◽  
Conclusive Evidence ◽  
Initial Contact ◽  
Single Exposure ◽  
Vessel Segment ◽  
Multiple Exposures ◽  
Wall Interaction ◽  
Platelet Spreading

SummaryIn order to explore further the mechanism by which glycoprotein GPIIb-IIIa promotes platelet vessel wall interaction, platelet adhesion to subendothelium was studied in an annular chamber in which subendothelium from rabbit aorta was exposed at a shear rate of 2,600 s−1 to blood from patients with thrombasthenia. Perfusions were conducted for each of 5 exposure times (1 ,2,3, 5 and 10 min), and the percent surface coverage of the vessel segment with platelets in the contact (C) and spread (S) stage was determined. Increased values of platelet contact (C) were obtained in thrombasthenia at all exposure times; this finding is consistent with a defect in platelet spreadirg, based on a previously described kinetic model of platelet attachment to subendothelium. According to this model of attachment, increased values of platelet contact (C) at a single exposure time may be indicative of either a defect in spreading (S) or initial contact (C), but multiple exposures will result in increased contact only for defects which are related to defectiye platelet spreading (s).The results obtained over a broad range of exposure times provide more conclusive evidence that GPIIb-IIIa mediates platelet spreading than those previously obtained at single exposure times.

Effects of Bupranolol, a New β-Blocker, on Platelet Functions of Rabbit and Human in Vitro

1976 ◽  
Vol 36 (02) ◽  
pp. 376-387 ◽  
Author(s):  
Teruhiko Umetsu ◽  
Kazuko Sanai ◽  
Tadakatsu Kato
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Rabbit Platelets ◽  
Β Blocker ◽  
Platelet Aggregates ◽  
Induced Aggregation ◽  
Platelet Functions

SummaryThe effects of bupranolol, a new β-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known β-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased.Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4–3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the β-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8–3.3 times as effectively as propranolol.From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.

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