title: PKC isoenzymes in equine platelets and stimulus induced activation.
abstract: Protein kinase C (PKC) is an important regulator of platelet activation and different isoenzymes can play positive and negative regulatory roles. The PKC isoenzymes expressed in equine platelets have not been documented but pharmacological inhibition has suggested a role for PKC delta (δ) in modulating responsiveness to platelet activating factor (PAF) (Brooks et al., 2009). Here the PKC isoenzyme profile in equine platelets has been characterised and PKCδ activation by PAF investigated. Platelet lysates were probed by Western blotting using a panel of antibodies against individual PKC isoenzymes. PKCδ and eight other isoenzymes were identified, namely classical PKCs alpha (α), beta (β), (both βI and βII) and gamma (γ), the novel PKCs epsilon (ɛ), eta (η) and theta (θ) and atypical PKC zeta (ζ). Having shown PKCδ to be present, a method was developed to measure PAF-induced isoenzyme translocation by preparing cytosolic and membrane fractions from digitonin permeabilised platelets. Phorbol 12-myristate 13-acetate (PMA) was shown to cause translocation of PKCδ to the membrane within 5s. PAF also caused PKCδ translocation although the response occurred more slowly; a significant, 7.6 ± 1.2 fold, increase in band density compared to unstimulated platelets was observed at 15 min; p=0.036, n=3. These data support a role for PKCδ in regulating PAF-induced functional responses in equine platelets.
authors.affiliation: Department of Veterinary Basic Sciences, Royal Veterinary College, Hawkshead Lane, Hertfordshire AL9 7TA, UK. iaktan@rvc.ac.uk
authors.forename: I
authors.initials: I
authors.lastname: Aktan
authors.forename: B
authors.initials: B
authors.lastname: Dunkel
authors.forename: F M
authors.initials: FM
authors.lastname: Cunningham
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006736:Horses; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000145:classification* / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D010972:Platelet Activating Factor; D011493:Protein Kinase C