Echicetin coated polystyrene beads: a novel tool to investigate GPIb-specific platelet activation and aggregation.
von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.
Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany.
Alexey
A
Navdaev
Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany.
Hariharan
H
Subramanian
Institute of Biophysics, Siberian Branch of the Russian Academy of Sciences, Krasnoyarsk, Russia.
Alexey
A
Petunin
Theodor Kocher Institute, University of Berne, Berne, Switzerland.
Kenneth J
KJ
Clemetson
Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany; Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia.
Stepan
S
Gambaryan
Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Mainz, Germany.
Ulrich
U
Walter
D001780:Blood Coagulation Tests / Q000379:methods; D002352:Carrier Proteins / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008863:Microspheres; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011137:Polystyrenes / Q000737:chemistry / Q000494:pharmacology*; D014757:Viper Venoms / Q000737:chemistry / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000494:pharmacology
D002352:Carrier Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D011137:Polystyrenes; D014757:Viper Venoms; D014841:von Willebrand Factor; C080995:echicetin