title: Pharmacologic protein kinase Cα inhibition uncouples human platelet-stimulated angiogenesis from collagen-induced aggregation.
abstract: Platelets promote angiogenesis by releasing angiogenesis-regulating factors from their α-granules upon aggregation. This effect has both physiologic and pathologic significance as it may contribute to carcinogenesis. Platelet α-granule release and aggregation are regulated, in part, via protein kinase C (PKC) α and β signaling. Our study investigated the effects of PKC inhibition on aggregation, angiogenesis-regulator secretion from α-granules, and platelet-stimulated angiogenesis. We hypothesized that selective PKCα inhibition may preferentially suppress angiogenesis-regulator secretion from α-granules but not aggregation, limiting platelet-stimulated angiogenesis. Human platelets were aggregated in the presence of conventional PKC inhibitors myr-FARKGALRQ and Ro 32-0432 (2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyridol[1,2-α]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide). Immunofluorescence microscopy of PKC translocation was used to determine the specificity of PKC-inhibitor targeting. Enzyme-linked immunosorbent assay was used to measure vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) release from platelets. Platelet effects on angiogenesis were tested using a capillary-formation assay. Ro 32-0432, but not the peptide inhibitor myr-FARKGALRQ (myristoylated-pseudosubstrate peptide inhibitor), inhibited aggregation in a concentration-dependent manner, while both Ro 32-0432 and myr-FARKGALRQ preferentially suppressed VEGF over TSP-1 secretion. Suppression of angiogenesis-regulator release occurred at inhibitor concentrations that did not significantly affect aggregation. Immunofluorescence microscopy revealed that PKCα targeting to α-granules is inhibited when angiogenesis-regulator secretion is uncoupled from aggregation. At concentrations that uncoupled α-granule release from aggregation, Ro 32-0432 and myr-FARKGALRQ inhibited platelet-stimulated angiogenesis. Hence, selective PKCα inhibition suppresses angiogenesis-regulator release from platelet α-granules with minimal effects on aggregation. Thus, selective PKCα inhibitors may have pharmacologic significance to regulate platelet-promoted angiogenesis.
authors: Faculty of Pharmacy and Pharmaceutical, University of Alberta, Edmonton, Alberta, Canada.
authors: Cesar
authors: C
authors: Moncada de la Rosa
authors: Aneta
authors: A
authors: Radziwon-Balicka
authors: Haitham
authors: H
authors: El-Sikhry
authors: John
authors: J
authors: Seubert
authors: Peter P
authors: PP
authors: Ruvolo
authors: Marek W
authors: MW
authors: Radomski
authors: Paul
authors: P
authors: Jurasz
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects*; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors*; D019700:Thrombospondin 1 / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; D004791:Enzyme Inhibitors; D019700:Thrombospondin 1; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D003094:Collagen; D051571:Protein Kinase C-alpha