Quantification of Platelet Adsesion in vivo

1979 ◽  
Author(s):  
H. Kortenhaus ◽  
G.V.R. Bom
Keyword(s):  
Fluorescence Microscopy ◽  
Small Proportion ◽  
Platelet Rich Plasma ◽  
Fluorescein Isothiocyanate ◽  
Cheek Pouch ◽  
Golden Hamsters ◽  
Cardiac Blood ◽  
Acid Citrate Dextrose ◽  
Citrate Dextrose

Golden hamsters (80-100g) were anaesthetised with pentobarbital and cardiac blood was collected into acid-citrate dextrose. Platelet-rich plasma brought to pH 6.2 was incubated at 37° with fluorescein-isothiocyanate (FITC: 100 μg/ml) for 10 min (Kortenhaus , Webelmann and Schroer: to be published). The platelets were injected i.v. into another hamster and observed in the cheek pouch circulation by fluorescence microscopy at x 500.In venules a small proportion of platelets were arrested, most for less than one sec., about 20% for up to 2 min and about 3% for longer. There was no correlation with rolling granulocyte frequencies (A therton & Born, 1972, Journal of Fhysiology, 222, 447).

Quantification of the adhesion of platelets in hamster venules in vivo

1982 ◽  
Vol 215 (1199) ◽  
pp. 135-145 ◽  
Keyword(s):  
Platelet Rich Plasma ◽  
Fluorescein Isothiocyanate ◽  
Adenosine Diphosphate ◽  
Cheek Pouch ◽  
Video Tape ◽  
Flow Through ◽  
Platelet Aggregates ◽  
The Mean ◽  
Adhesion Of Platelets

(i) Citrated platelet-rich plasma freshly prepared from golden hamsters was mixed with fluorescein isothiocyanate (FITC) which made the platelets fluorescent. These platelets were injected intravenously into anaesthetized hamsters with exteriorized cheek pouch preparations superfused at 37 °C with Krebs-bicarbonate solution. The exposed microcirculation was observed microscopically by bright field or fluores­cent illumination. The flowing and sticking of fluorescent platelets was recorded on video tape for quantitative analysis. (ii) In four experiments 22–36%, mean 28%, of fluorescent platelets were circulating 2-3 h after their injection. In seven experiments the fluorescent platelets accounted for 0.6–3.3 %, mean 1.7 %, of circulating platelets. (iii) In venules 20–60 μm in diameter small proportions, mean 5.4%, of circulating fluorescent platelets stopped moving by sticking to the vessel walls. About 80 % of these platelets stuck for up to 1 s, a further 10-15% for up to 5 s, and only about 2% for longer than 2 min. There was an inverse relation between size of venule and proportion of platelets sticking in them. (iv) There was a direct relation between the mean velocities at which platelets flowed through the venules and the sizes of the venules. In the smaller venules the velocity distribution of the platelets had a clear maximum which was not as evident in larger venules. (v) In a few observations on arterioles, flowing platelets could not be seen, and arrested platelets only in a dilatation and at a capillary branch. (vi) Ethylenediamine tetraacetate in the superfusing fluid decreased platelet sticking in venules but did not abolish it. (vii) Adenosine diphosphate in the superfusing fluid caused the appear­ance of platelet aggregates in venules and of sticking platelets in arterioles during progressive diminution in blood flow through both types of vessel. (viii) The observations make it improbable that the release of platelet constituents affects normal venules or arterioles except, possibly, where haemodynamic conditions are affected by wall irregularities such as dilations or branching.

scholarly journals A Comparative Study of the Effects of Anticoagulants on Pure Platelet-Rich Plasma Quality and Potency

Biomedicines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 42 ◽  
Author(s):  
Hachidai Aizawa ◽  
Hideo Kawabata ◽  
Atsushi Sato ◽  
Hideo Masuki ◽  
Taisuke Watanabe ◽  
...  
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Platelet Rich Plasma ◽  
In Vivo Studies ◽  
Negative Effects ◽  
Acid Citrate Dextrose ◽  
Citrate Dextrose ◽  
High Level ◽  
Activation Status

It is generally accepted that citrate or the A-form of acid-citrate-dextrose (ACD-A) are suitable for preparing platelet-rich plasma (PRP) for regenerative therapy. However, this is based on evidence from blood transfusions and not from regenerative medicine. Thus, we examined the effects of anticoagulants, such as ACD-A, ethylenediaminetetraacetic acid (EDTA), and heparin, on the regenerative quality of PRP to address this gap. The blood samples were collected in the presence of anticoagulants and were processed to prepare pure-PRP. Platelet size, activation status, and intra-platelet free Ca2+ concentration were determined while using a hematology analyzer and flow cytometer. Platelet-derived growth factor-BB (PDGF-BB) was quantified while using an ELISA. In pure-PRP samples, EDTA caused platelet swelling and activation, but yielded the highest number of platelets. Heparin aggregated platelets and disturbed the overall counting of blood cells. However, no significant differences in PDGF-BB levels were observed among the anticoagulants tested. Moreover, when considering the easy preparation of platelet suspensions, without the need for high-level pipetting skills, these findings suggest the comparable potency of EDTA-derived pure-PRP in tissue regeneration and support the use of EDTA in the preparation of pure-PRP. Further in vivo studies are required in animal models to exclude the possible negative effects of including EDTA in pure-PRP preparations.

Conditions Affecting the Responses of Human Platelets to Epinephrine

1988 ◽  
Vol 60 (02) ◽  
pp. 209-216 ◽  
Author(s):  
Chantal Lalau Keraly ◽  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
Hidenori Suzuki ◽  
J Fraser Mustard
Keyword(s):  
Platelet Rich Plasma ◽  
Shape Change ◽  
Human Platelets ◽  
Dense Granule ◽  
Primary Phase ◽  
Lag Phase ◽  
Acid Citrate Dextrose ◽  
Two Phases ◽  
Citrate Dextrose ◽  
Thromboxane Formation

SummaryConditions affecting the responses of human platelets to epinephrine were examined. In platelet-rich plasma prepared from blood anticoagulated with hirudin or PPACK (D-pheny- lalanyl-L-prolyl-L-arginine chloromethyl ketone), epinephrine did not cause shape change or aggregation. In a Tyrode-albumin- apyrase solution containing a concentration of Ca2+ in the physiological range, and fibrinogen, epinephrine in concentrations as high as 40 μM did not induce platelet shape change, caused either no primary aggregation or very slight primary aggregation, and did not induce thromboxane formation, release of dense granule contents, or secondary aggregation. In contrast, in citrated platelet-rich plasma, epinephrine induced two phases of aggregation. This is not attributable to the generation of traces of thrombin since the same effects were evident when blood was taken into a combined citrate-hirudin anticoagulant or a combined citrate-PPACK anticoagulant. In a modified Tyrode-albu- min-apyrase solution containing approximately 20 μM Ca2+, 1 mM Mg2+, and fibrinogen, epinephrine induced extensive aggregation after a lag phase, but no primary phase was evident; thromboxane formation and release of dense granule contents accompanied the aggregation response. These responses were also observed when PPACK was included with the acid-citrate- dextrose anticoagulant, and in the washing and resuspending fluids. In the presence of aspirin or the thromboxane receptor blocker BM 13.177 a few small aggregates were detected by particle counting and by scanning electron microscopy; with the latter inhibitor, the platelets in the aggregates retained their disc shape; secondary aggregation and the responses associated with it did not occur. Thus thromboxane A2 formation is not necessary for the formation of these small aggregates, but is required for extensive aggregation and release. As with other weak agonists, the close platelet-to-platelet contact in the low Ca2+ medium appears to be necessary for full secondary aggregation. Omission of fibrinogen from the low Ca2+ medium prevented both primary and secondary aggregation in response to epinephrine. An antibody (10E5) to the glycoprotein Ilb/IIIa complex was completely inhibitory in the presence of fibrinogen. Thus the response of human platelets to epinephrine is influenced by the concentration of Ca2+ and the presence of fibrinogen in the medium in which they are suspended.

Tannic acid elicits neurogenic plasma exudation from the in situ hamster cheek pouch

1998 ◽  
Vol 274 (1) ◽  
pp. R237-R242
Author(s):  
Xiao-Pei Gao
Keyword(s):  
Tannic Acid ◽  
Biosynthetic Pathway ◽  
Fluorescein Isothiocyanate ◽  
Cheek Pouch ◽  
Site Formation ◽  
Hamster Cheek Pouch ◽  
Plasma Exudation ◽  
Synthase Inhibitor

The purpose of this study was to determine whether tannic acid elicits neurogenic plasma exudation from the oral mucosa in vivo and, if so, whether this response is transduced in part by thel-arginine-nitric oxide (NO) biosynthetic pathway. Using intravital microscopy, we found that suffusion of tannic acid elicits significant concentration-dependent leaky site formation and increase in clearance of fluorescein isothiocyanate-dextran (molecular mass 70 kDa) from the in situ hamster cheek pouch ( P < 0.05). These effects are significantly attenuated by two selective, but structurally distinct, nonpeptide neurokinin-1 (NK1) receptor antagonists, CP-96,345 and RP-67580, but not by CP-96,344, the 2R,3R enantiomer of CP-96,345. N G-nitrol-arginine methyl ester (l-NAME), an NO synthase inhibitor, but notd-NAME, significantly attenuates tannic acid-induced responses.l-Arginine, but notd-arginine, reverses the attenuating effects of l-NAME. We conclude that tannic acid elicitsl-arginine-NO biosynthetic pathway-dependent neurogenic plasma exudation from the in situ hamster cheek pouch.

scholarly journals Dexamethasone attenuates acute macromolecular efflux increase evoked by smokeless tobacco extract

1999 ◽  
Vol 87 (2) ◽  
pp. 619-625 ◽  
Author(s):  
Xiao-Pei Gao ◽  
Syed R. Akhter ◽  
Hiroyuki Ikezaki ◽  
Dennis Hong ◽  
Israel Rubinstein
Keyword(s):  
Oral Mucosa ◽  
Smokeless Tobacco ◽  
Fluorescein Isothiocyanate ◽  
Cheek Pouch ◽  
Site Formation ◽  
Hamster Cheek Pouch ◽  
Acute Increase ◽  
Tobacco Extract

The purpose of this study was to determine whether dexamethasone attenuates the acute increase in macromolecular efflux from the oral mucosa elicited by an aqueous extract of smokeless tobacco (STE) in vivo, and, if so, whether this response is specific. Using intravital microscopy, we found that 20-min suffusion of STE elicited significant, concentration-related leaky site formation and an increase in clearance of fluorescein isothiocyanate-labeled dextran (FITC-dextran; mol mass 70 kDa) from the in situ hamster cheek pouch ( P < 0.05). This response was significantly attenuated by dexamethasone (10 mg/kg iv). Dexamethasone also attenuated the bradykinin-induced leaky site formation and the increase in clearance of FITC-dextran from the cheek pouch. However, it had no significant effects on adenosine-induced responses. Dexamethasone had no significant effects on baseline arteriolar diameter and on bradykinin-induced vasodilation in the cheek pouch. Collectively, these data indicate that dexamethasone attenuates, in a specific fashion, the acute increase in macromolecular efflux from the in situ oral mucosa evoked by short-term suffusion of STE. We suggest that corticosteroids mitigate acute oral mucosa inflammation elicited by smokeless tobacco.

Nitric oxide accounts for histamine-induced increases in macromolecular extravasation

1994 ◽  
Vol 266 (6) ◽  
pp. H2369-H2373 ◽  
Author(s):  
W. G. Mayhan
Keyword(s):  
Nitric Oxide ◽  
Fluorescent Microscopy ◽  
Fluorescein Isothiocyanate ◽  
Cheek Pouch ◽  
Hamster Cheek Pouch ◽  
Before And After ◽  
Subsequent Activation ◽  
Ly 83583

The goal of this study was to determine the role of nitric oxide in histamine-induced increases in macromolecular extravasation in the hamster cheek pouch in vivo. We used intravital fluorescent microscopy and fluorescein isothiocyanate dextran (FITC-dextran; mol wt = 70,000 K) to examine extravasation from postcapillary venules in response to histamine before and after application of an enzymatic inhibitor of nitric oxide, NG-monomethyl-L-arginine (L-NMMA; 1.0 microM). Increases in extravasation of macromolecules were quantitated counting the number of venular leaky sites. Histamine (1.0 and 5.0 microM) increased the number of venular leaky sites from zero (basal conditions) to 11 +/- 1 and 21 +/- 2/0.11 cm2, respectively. Superfusion of L-NMMA (1.0 microM) and LY-83583 (1.0 microM) significantly decreased histamine-induced formation of venular leaky sites, whereas L-arginine (100 microM) potentiated histamine-induced formation of venular leaky sites. In contrast, superfusion of NG-monomethyl-D-arginine (1.0 microM) did not inhibit the formation of venular leaky sites in response to histamine. Thus the findings of the present study suggest that production of nitric oxide, and subsequent activation of guanylate cyclase, plays an important role in macromolecular efflux in vivo in response to histamine.

The Effect of Methylprednisolone Sodium Succinate on Erythrocyte and Haemoglobin Function

1980 ◽  
Vol 59 (3) ◽  
pp. 163-168 ◽  
Author(s):  
M. Brada ◽  
L. A. Robinson ◽  
A. J. Bellingham
Keyword(s):  
Whole Blood ◽  
Sodium Succinate ◽  
Oxygen Affinity ◽  
Whole Cells ◽  
Methylprednisolone Sodium Succinate ◽  
Acid Citrate Dextrose ◽  
Citrate Dextrose ◽  
Therapeutic Doses

1. As it has been suggested that the beneficial effect of methylprednisolone in shock is due to its effect on erythrocyte oxygen affinity, we studied its effect on incubated erythrocytes and on haemoglobin solution. 2. Incubation of fresh whole blood anticoagulated with acid/citrate/dextrose with methylprednisolone (7 mmol/l) produced a significant decrease in oxygen affinity, which was not seen with lower concentrations of methylprednisolone. When either acid/citrate/dextrose blood stored for 10 days or fresh heparinized blood was used, no significant increase in the partial pressure of oxygen at 50% haemoglobin saturation (P50) was demonstrated even with methylprednisolone at 7 mmol/l. At the highest concentration achieved in plasma with standard therapeutic doses (56 μmol/l) there was no increase in P50 under all the conditions studied. 3. Methylprednisolone reduced the oxygen affinity of haemoglobin in solution. The reduction in oxygen affinity was less than that produced by 2,3-diphosphoglycerate and more than that of either sodium succinate or sodium chloride. 4. From the results of this study we conclude that the effect observed in whole cells is probably due to a direct effect of methylprednisolone on haemoglobin. To produce a significant decrease of oxygen affinity of whole blood in vitro requires a plasma concentration of methylprednisolone above that obtained in plasma in vivo, with the currently used therapeutic doses.

Effect of Adenosine on Phosphofructokinase Activity of Preserved Blood

1957 ◽  
Vol 189 (1) ◽  
pp. 105-107 ◽  
Author(s):  
Y. S. Brownstone ◽  
M. C. Blanchaer
Keyword(s):  
Protective Effect ◽  
Adenosine Triphosphate ◽  
Glycolytic Enzyme ◽  
Red Cells ◽  
Progressive Decrease ◽  
Beneficial Effects ◽  
Phosphofructokinase Activity ◽  
Acid Citrate Dextrose ◽  
Citrate Dextrose

Adenosine in concentrations of 5–20 mm prevents the progressive decrease in activity of the erythrocyte glycolytic enzyme phosphofructokinase in blood preserved in acid citrate-dextrose at 4°. In hemolysates the protective effect of adenosine is also exhibited by its metabolic derivatives inosine, ribose-5-phosphate and adenosine triphosphate. The beneficial effects of adenosine on in vivo survival of stored red cells, glycolysis and PFK activity suggests that the integrity of this enzyme may influence the keeping qualities of ACD preserved blood.

Cigarette smoke extract potentiates bradykinin-induced increases in microvascular permeability

1993 ◽  
Vol 75 (1) ◽  
pp. 27-32 ◽  
Author(s):  
W. G. Mayhan ◽  
I. Rubinstein
Keyword(s):  
Cigarette Smoke ◽  
Fluorescein Isothiocyanate ◽  
Cigarette Smoke Extract ◽  
Microvascular Permeability ◽  
Cheek Pouch ◽  
Site Formation ◽  
Hamster Cheek Pouch ◽  
Fluorescein Isothiocyanate Dextran ◽  
Macromolecular Permeability

The first goal of this study was to determine whether cigarette smoke extract (CSE) increases microvascular permeability of the hamster cheek pouch in vivo. The second goal was to determine whether CSE potentiates bradykinin-induced increases in vascular permeability in the hamster cheek pouch. Using intravital microscopy, we examined the permeability of the hamster cheek pouch to fluorescein isothiocyanate-dextran (mol wt 70,000). Increases in permeability were quantitated by counting the number of postcapillary venular leaky sites per 0.11 cm2. Superfusion of CSE (1, 5, and 10%) did not produce venular leaky sites and, thus, did not alter macromolecular permeability. Superfusion of bradykinin (0.1, 0.5, and 1.0 microM) produced a dose-related increase in the number of venular leaky sites. Formation of leaky sites in response to bradykinin was potentiated by CSE. To determine whether potentiation of bradykinin-induced leaky site formation by CSE was related to products released via the cyclooxygenase pathway, we examined the effects of pretreatment with indomethacin (10 mg/kg i.v.). Indomethacin did not alter the potentiating effect of CSE on bradykinin-induced leaky site formation. These findings suggest that CSE does not alter basal permeability of the hamster cheek pouch microcirculation in vivo. However, CSE potentiates bradykinin-induced increases in microvascular permeability. The mechanism of CSE-induced potentiation of microvascular permeability does not appear to be related to substances produced via the cyclooxygenase pathway.

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