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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.

SUBMITTER: Marshall SJ

PROVIDER: S-EPMC1222309 | biostudies-other | 2002 Jan

REPOSITORIES: biostudies-other

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Project description:Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I2 (PGI2), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 microM) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840 +/- 8920 compared with 81050 +/- 6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI2. To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha-thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C PKC) activation after 10 min stimulation compared with alpha-thrombin. These concentrations also suppressed PGI2-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI2-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentration of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 microM) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI2-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, Gi. We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and Gi, as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI2 to raise cyclic AMP concentration from 1.4 +/- 0.7 to 4.1 +/- 1.3 nmol/10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules.

Project description:The major platelet integrin, glycoprotein IIb-IIIa, binds soluble fibrinogen only after platelet activation. To investigate the mechanism by which platelets convert glycoprotein IIb-IIIa into a functional fibrinogen receptor, we characterized the opening and closing of fibrinogen-binding sites in isolated platelet membranes and compared the regulatory properties of membrane-bound glycoprotein IIb-IIIa with those of the detergent-solubilized receptor. Basal fibrinogen binding to the membranes possessed many of the properties of fibrinogen binding to activated platelets; however, less than 10% of glycoprotein IIb-IIIa in the membranes was capable of binding fibrinogen. Preincubating the membranes with either an activating glycoprotein IIb-IIIa antibody or alpha-chymotrypsin increased fibrinogen binding. In contrast, agents that require intracellular mediators, such as platelet agonists, guanine-nucleotide-binding-protein activators and purified protein kinase C, did not stimulate fibrinogen binding to the membranes, suggesting that cytosolic factor(s) may be required for activation of the receptor in platelets. Occupancy of glycoprotein IIb-IIIa in the membranes with RGD (Arg-Gly-Asp)-containing peptides reversibly exposed neoantigenic epitopes and fibrinogen-binding sites in the receptor. These conformational changes required membrane fixation to be maintained following peptide removal. Similar results were obtained with purified glycoprotein IIb-IIIa incorporated into phospholipid vesicles, indicating that the resting state of the receptor is favoured in these environments. In contrast, when the conformation of detergent-solubilized glycoprotein IIb-IIIa was altered by exposure to RGD-containing peptides, the receptor remained active even after incorporation into phospholipid vesicles. These results demonstrate that platelet membranes are a useful model in which to study the regulation of glycoprotein IIb-IIIa and suggest that the environment surrounding the receptor may have a profound influence on this process.

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Glycoprotein IIb-IIIa-dependent aggregation by glycoprotein Ibalpha is reinforced by a Src family kinase inhibitor (PP1)-sensitive signalling pathway.

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