title: Glycoprotein IIb-IIIa-dependent aggregation by glycoprotein Ibalpha is reinforced by a Src family kinase inhibitor (PP1)-sensitive signalling pathway.
abstract: It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) gamma-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR gamma-chain, Syk and PLCgamma2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCgamma2 nor functional responses, such as Ca(2+) elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbalpha in washed platelets does not promote functional responses.
authors.affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK. stuart.marshall@pharm.ox.ac.uk
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Marshall
authors.forename: Naoki
authors.initials: N
authors.lastname: Asazuma
authors.forename: Denise
authors.initials: D
authors.lastname: Best
authors.forename: Peter
authors.initials: P
authors.lastname: Wonerow
authors.forename: Gary
authors.initials: G
authors.lastname: Salmon
authors.forename: Robert K
authors.initials: RK
authors.lastname: Andrews
authors.forename: Steve P
authors.initials: SP
authors.lastname: Watson
mesh_terms: D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D010766:Phosphorylation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D011961:Receptors, Fc / Q000378:metabolism; D000072377:Syk Kinase; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors*
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011720:Pyrazoles; D011743:Pyrimidines; D011961:Receptors, Fc; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D019061:src-Family Kinases; D010738:Type C Phospholipases