Biomaterials, Artificial Cells and Immobilization Biotechnology
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Effect of a High Concentration PERFLUOCARBON Emulsion on Platelet Function Dan J. Smith & Thomas A. Lane To cite this article: Dan J. Smith & Thomas A. Lane (1992) Effect of a High Concentration PERFLUOCARBON Emulsion on Platelet Function, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 1045-1049, DOI: 10.3109/10731199209119761 To link to this article: http://dx.doi.org/10.3109/10731199209119761
Published online: 11 Jul 2009.
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [University of Cincinnati Libraries]
Date: 22 March 2016, At: 21:26
ART.
CELLS & IMMOB.
BIOTECH.,
Z O ( 2 - 4 ) , 1045-1049 ( 1 9 9 2 )
Downloaded by [University of Cincinnati Libraries] at 21:26 22 March 2016
BIOMAT.,
1045
Copyright 0 1992 by Marcel Dekker, Inc.
1046
SMITH AND LANE
Downloaded by [University of Cincinnati Libraries] at 21:26 22 March 2016
MATERIALS AND METHODS 1. Ex-vivo porcine platelet aggregation: Prior to infusion of perflubron emulsions, and at 10 and 120 minutes after perflubron infusion, platelet rich plasma (PRP) was prepared from cilrdted pig blood. Platelet counts were adjusted to 3.0 X 10 '/miwllh autologous platelet-poor plasma. Control and perflubron emulsions were infused, generally at 3 mYKg (0,2ml/kg/min) unless otherwise noted. Agonists. with final concenuations in parentheses. used to stimulate aggregation were collagen (6mgIml). ADP (20mM) and AA (3.2mM). Platelet aggregation was measured by the photo optical method and percent aggregation was quantitated. 2. In V i m Human Platelet Calcium Rux: A flow cytomeuic assay has been developed which uses the CP2 chelator indo-1 to quantitate calcium flux. When indo-1 is excited with an ultraviolet laser at 380 nM, a shift in emission specua occurs from 480 nM for the unbound Indo-1 to 400 nM for the Ca r 2 -bound-Indo-1complex. The resulting ratio of emission spectra (400 nW480nM signal) is a function of Ca *2 levels. We used this methodology to quantitate agonist-induced platelet activation pre and post in v i m treatment of human platelets with perflubron, FluosoL and Inrralipid. Platelet rich plasma was obtained from hepainized human blood. Platelets were loaded with Indo-1 and flow cytometric analysis was carried out. Parameters noted were forward angle light scatter and 90 degree light scatter (loose indications of size and granularity, respectively). We also measured platelet numbers and the bound vs unbound Indo-1 ratio as a function of time. The platelet agonists ADP and arachidonic acid along with the Ca+2ionophore A23 187 were used to stimulate the platelets pre and post treatment with the emulsions. The Ca*? flux experiments were divided into four types. 1) Assessment of direct activation by perflubron: The treatment (pemubron. Fl/tosol or Infrolipid) was added to the Indo-1 loaded platelets and change in Cat' levels was measured. 2) Effect of perflubron on agonist-induced Cai2 level: If no enhancement of Ca" level was seen post addition of emulsions, calcium ionophore, (A23187) or a platelet agonist (ADP or AA) was added to the Indo-1 loaded plateleUemulsion mixture at a near-maximal stirnulatory dose. The platelet C b 2 level was compared to that of the non-treated platelets and any inhibition was reported. 3) Effect of emulsion on agonist dose response: if the treated platelets showed inhibited Cat' flux or levels, then the dose of treatment was held constant, but the stimulant dose was increased in an attempt to overcome the inhibitory effect. 4) Emulsion dose response: In some cases, the emulsion concentration was varied to see if the treatment dose effected the inhibition of stimulant-induced platelet Ca+2level. The data is reported as percent of platelets responding to the stimulant being tested, the percent cells which are stimulated to greater than rlireshold, and mean Indo-1 ratio of cells after stimulation. The "percent cells responding" is the percent of platelels which have a Ca'2 bound Indo-1 ratio above that of the resting or unstimulated cell population. The "percent greater than threshold is the proponion of platelets responding above a chosen threshold level with time. In this case, the threshold value is set at a level at which c 5% of resting cells are above. This value is a rough parameter of cell heterogeneity. A fluorescence "quenching" experiment was also mn to insure that the emulsions were not decreasing or "quenching" the fluorescent signal emitted by the Indo-1 loaded platelets. The signal was not found to be quenched by the emulsions
RESULTS 1. Platelet Aggregation: T-I0 min and T-120 min data arc reported :is percents. relative to the aggregation of the T = 0 sample which was set at 100%.
a. Saline, (n = 2) 3 ml/kg. 0.2 mlikglmin; Agonist Collagen ADP AA
Mean Data: Relative to T = 0 Sample
'I= 10
T = 120
125% 105% 100%
90% 100% 100%
b. Flrrosol-DA, (n = 4). 3 mlkg. 0.2 ml/kg/min or 15 mlkg. 1.0
ml/ks/min.
Mean Data: Relative to T = 0 Sample Agonist
T - 10
T = 120
Collagen ADP
115%
100% 88% 82%
AA
76% 78%
1047
EFFECT OF PFC EMULSION ON PLATELET FUNCTION 'lAlll.li I' Prc 1'rc:itcd l ' l : i ~ c l c t ~Stiinul:itcd with 250 IIM A23187 'h > 'l~lire!;liold 99 x 20 6 12.0 91 2
%, I 'l'hrcsl~~~ld
% I
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Effect of a High Concentration PERFLUOCARBON Emulsion on Platelet Function Dan J. Smith & Thomas A. Lane To cite this article: Dan J. Smith & Thomas A. Lane (1992) Effect of a High Concentration PERFLUOCARBON Emulsion on Platelet Function, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 1045-1049, DOI: 10.3109/10731199209119761 To link to this article: http://dx.doi.org/10.3109/10731199209119761
Published online: 11 Jul 2009.
Submit your article to this journal
Article views: 3
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [University of Cincinnati Libraries]
Date: 22 March 2016, At: 21:26
ART.
CELLS & IMMOB.
BIOTECH.,
Z O ( 2 - 4 ) , 1045-1049 ( 1 9 9 2 )
Downloaded by [University of Cincinnati Libraries] at 21:26 22 March 2016
BIOMAT.,
1045
Copyright 0 1992 by Marcel Dekker, Inc.
1046
SMITH AND LANE
Downloaded by [University of Cincinnati Libraries] at 21:26 22 March 2016
MATERIALS AND METHODS 1. Ex-vivo porcine platelet aggregation: Prior to infusion of perflubron emulsions, and at 10 and 120 minutes after perflubron infusion, platelet rich plasma (PRP) was prepared from cilrdted pig blood. Platelet counts were adjusted to 3.0 X 10 '/miwllh autologous platelet-poor plasma. Control and perflubron emulsions were infused, generally at 3 mYKg (0,2ml/kg/min) unless otherwise noted. Agonists. with final concenuations in parentheses. used to stimulate aggregation were collagen (6mgIml). ADP (20mM) and AA (3.2mM). Platelet aggregation was measured by the photo optical method and percent aggregation was quantitated. 2. In V i m Human Platelet Calcium Rux: A flow cytomeuic assay has been developed which uses the CP2 chelator indo-1 to quantitate calcium flux. When indo-1 is excited with an ultraviolet laser at 380 nM, a shift in emission specua occurs from 480 nM for the unbound Indo-1 to 400 nM for the Ca r 2 -bound-Indo-1complex. The resulting ratio of emission spectra (400 nW480nM signal) is a function of Ca *2 levels. We used this methodology to quantitate agonist-induced platelet activation pre and post in v i m treatment of human platelets with perflubron, FluosoL and Inrralipid. Platelet rich plasma was obtained from hepainized human blood. Platelets were loaded with Indo-1 and flow cytometric analysis was carried out. Parameters noted were forward angle light scatter and 90 degree light scatter (loose indications of size and granularity, respectively). We also measured platelet numbers and the bound vs unbound Indo-1 ratio as a function of time. The platelet agonists ADP and arachidonic acid along with the Ca+2ionophore A23 187 were used to stimulate the platelets pre and post treatment with the emulsions. The Ca*? flux experiments were divided into four types. 1) Assessment of direct activation by perflubron: The treatment (pemubron. Fl/tosol or Infrolipid) was added to the Indo-1 loaded platelets and change in Cat' levels was measured. 2) Effect of perflubron on agonist-induced Cai2 level: If no enhancement of Ca" level was seen post addition of emulsions, calcium ionophore, (A23187) or a platelet agonist (ADP or AA) was added to the Indo-1 loaded plateleUemulsion mixture at a near-maximal stirnulatory dose. The platelet C b 2 level was compared to that of the non-treated platelets and any inhibition was reported. 3) Effect of emulsion on agonist dose response: if the treated platelets showed inhibited Cat' flux or levels, then the dose of treatment was held constant, but the stimulant dose was increased in an attempt to overcome the inhibitory effect. 4) Emulsion dose response: In some cases, the emulsion concentration was varied to see if the treatment dose effected the inhibition of stimulant-induced platelet Ca+2level. The data is reported as percent of platelets responding to the stimulant being tested, the percent cells which are stimulated to greater than rlireshold, and mean Indo-1 ratio of cells after stimulation. The "percent cells responding" is the percent of platelels which have a Ca'2 bound Indo-1 ratio above that of the resting or unstimulated cell population. The "percent greater than threshold is the proponion of platelets responding above a chosen threshold level with time. In this case, the threshold value is set at a level at which c 5% of resting cells are above. This value is a rough parameter of cell heterogeneity. A fluorescence "quenching" experiment was also mn to insure that the emulsions were not decreasing or "quenching" the fluorescent signal emitted by the Indo-1 loaded platelets. The signal was not found to be quenched by the emulsions
RESULTS 1. Platelet Aggregation: T-I0 min and T-120 min data arc reported :is percents. relative to the aggregation of the T = 0 sample which was set at 100%.
a. Saline, (n = 2) 3 ml/kg. 0.2 mlikglmin; Agonist Collagen ADP AA
Mean Data: Relative to T = 0 Sample
'I= 10
T = 120
125% 105% 100%
90% 100% 100%
b. Flrrosol-DA, (n = 4). 3 mlkg. 0.2 ml/kg/min or 15 mlkg. 1.0
ml/ks/min.
Mean Data: Relative to T = 0 Sample Agonist
T - 10
T = 120
Collagen ADP
115%
100% 88% 82%
AA
76% 78%
1047
EFFECT OF PFC EMULSION ON PLATELET FUNCTION 'lAlll.li I' Prc 1'rc:itcd l ' l : i ~ c l c t ~Stiinul:itcd with 250 IIM A23187 'h > 'l~lire!;liold 99 x 20 6 12.0 91 2
%, I 'l'hrcsl~~~ld
% I
