C. M. Edwards

Pluronid® F-68 Inhibits Agonist-Induced Platelet Aggregation in Human Whole Blood in Vitro

The effects have been studied of Pluronic F-68 at 0.04% (w/v) on platelet aggregation in hirudin (50 micrograms ml-1)-anticoagulated, human whole blood in vitro in response to the following aggregation agonists: (i) phorbol 12-myristate 13-acetate (PMA; 0.05, 0.1 or 0.15 microgram ml-1), (ii) collagen (0.125, 0.25 or 0.5 microgram ml-1), or (iii) ristocetin (0.3, 0.6 or 1.2 micrograms ml-1). Pluronic F-68 significantly (P < 0.05) inhibited platelet aggregation that followed the addition of all agonists at their lowest concentration tested. Pluronic F-68 had markedly less pronounced inhibitory effects on the platelet aggregation that occurred in response to 0.15 microgram ml-1 PMA, where the mean % aggregation after 8 min was 67% of control (P < 0.05). Pluronic F-68 did not alter platelet aggregation in blood treated with 0.25 or 0.5 microgram ml-1 of collagen.

Effects of a Novel Perfluorocarbon Emulsion on Neutrophil Chemiluminescence in Human Whole Blood in Vitro

The effects have been studied of a novel perfluorochemical (PFC) emulsion (18.5% perfluorodecalin, 1.5% perfluorodimorpholine propane, 2.5% lecithin) on phorbol 12-myristate 13-acetate (PMA; 100 micrograms ml-1)-induced neutrophil chemiluminescence in citrated human whole blood in vitro. A transient, dose-dependent, decrease in chemiluminescence, to a maximum of 54% after 12 min (P < 0.05), occurred when blood was pre-incubated with 10-40 microliters of the PFC emulsion, compared to saline controls. The mean (+/- s.e.m., n = 6) chemiluminescence of neutrophils incubated with 30 microliters emulsion at 12 min following PMA stimulation (9.5 +/- 1.3 mV) was significantly lower (P < 0.05) than control (24.2 +/- 2.2 mV). Incubation of blood with lecithin up to 16 mg ml-1 and Pluronic F-68 or Pluronic PE 6800 up to 65 mg ml-1 did not affect chemiluminescence.

Changes in Cell Biochemistry in Response to Culture of Protoplasts with Oxygenated Perfluorocarbon

Superoxide dismutase (superoxide oxidoreductase; EC 1.15.1.1; SOD) was measured in enzymatically isolated protoplasts of Salpiglossis sinuata following culture in aqueous nutrient medium overlaying oxygen-gassed perfluorodecalin (Flutec PP6; BNFL Fluorochemicals, UK). SOD was extracted from harvested, lysed protoplast-derived cells after 1, 3, 7 and 14 days of culture and assayed spectrophotometrically. Protoplasts cultured with oxygenated PFC (+/- s.e.m, n = 5) showed significant increases in mean SOD activity to 4.2 +/- 0.1 U after 1 day (P < 0.05) and 9.3 +/- 0.7 U after 3 days (P < 0.01), with a fall in mean SOD after 7 days (5.1 +/- 0.9 U), similar to control. The decrease in SOD after 7 days correlated closely with a progressive fall in pO2 in the PFC phase over the same period. In contrast, control protoplasts (medium alone) or protoplasts cultured in medium overlaying non-oxygenated PFC showed no significant changes in mean SOD activity over the 14-day culture assessment period.

Effects of the co-polymer surfactant, Pluronic® F-68, on platelet aggregation in human whole blood

The effects have been studied of Pluronic F-68 on platelet aggregation in human whole blood. The median spontaneous platelet aggregation in normal blood (n = 15) was 18.4% [interquartile range (IQ) = 10.5-24.2%]. Commercial grade Pluronic F-68 significantly (P < 0.05) reduced platelet aggregation at 7.3 microM (median = 8.4%, IQ = 3.9-13.4; n = 12) and almost eliminated aggregation at concentrations of > 58 microM (median = 2.0%, IQ = 0.0-3.5). Similar results were obtained with a silica gelpurified Pluronic F-68 fraction (n = 3). Pluronic F-68 also accelerated the rate of platelet dis-aggregation in blood treated with 0.3, 1.0 or 3.0 mM adenosine di-phosphate. These results suggest that the therapeutic effects of Pluronic F-68 in ischaemic injury may be due, in part, to inhibition of platelet aggregation in the microcirculation. The beneficial effects of tissue perfusion with oxygen-carrying perfluorochemical emulsions, containing Pluronic F-68, may also involve direct effects of the surfactant on platelets.

Evaluation of commercial and purified Pluronic F-68 in a human blood neutrophil bioassay.

The effects have been studied of commercial grade Pluronic F-68 or its purified fractions, prepared by passage through silica gel resin (SGR) or by supercritical fluid fractionation (SFF), on human polymorphonuclear leucocyte (PMNL) chemiluminescence in vitro. The mean (+/- s.d., n = 3) total chemiluminescence following stimulation of neutrophils with phorbol 12-myristate 13-acetate in saline controls, was 190 +/- 3 mV x min. Commercial Pluronic inhibited chemiluminescence by a maximum of 26% (P < 0.05), whilst, in contrast, Pluronic F-68 fractions prepared by SGR or SFF stimulated chemiluminescence by up to 53% over control (P < 0.05). The total chemiluminescence with Pluronic F-68 prepared by SFF followed by SGR was not significantly different to that produced by saline (0.9% w/v NaCl). These results reinforce previous suggestions that trace impurities in commercial preparations of the Pluronic F-68 are responsible for reported adverse biological effects.

Novel Fluorosurfactants For Perfluorochemical Emulsification

Liquid perfluorochemicals (PFC) are highly inert, can dissolve large volumes of respiratory gases and have been administered intravascularly as emulsions. One such “firstgeneration” ommercial emulsion, Fluosol® (Alpha Therapeutic, U.K.), has been approved for clinical use as an adjunct to percutaneous transluminal coronary angioplasty (Lowe, 1994a,b). Current research is aimed at the development of more concentrated “second-” and “third-generation” formulations with improved stability and oxygen-transport properties. In this context, there is increasing interest in the use of tailor-made, “fluorophilic” surfactants, either as sole or co-emulsifiers, to enhance PFC emulsion stability (Kissa, 1993; Riess and Greiner, 1993). Ideally, such surfactants should consist of a hydrophobic moiety, which interacts with the PFC (disperse) phase, and a hydrophilic end, which interacts with the aqueous (continuous) phase. Such compounds reduce the interfacial tension between the two phases, thus facilitating emulsification and contributing to emulsion stability. In addition to their specific chemical features, fluoro-surfactants for PFC emulsification must have acceptable biocompatibility characteristics, particularly relating to their interactions with blood components.