Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5'-[gamma-thio]triphosphate and by thrombin in the presence of GTP

• Published in: Biochemical journal Vol. 243; no. 2; pp. 457 - 465
• Main Authors: Hrbolich, J K, Culty, M, Haslam, R J
• Format: Journal Article
• Language: English
• Published: England 15.04.1987
• Subjects: Adenosine Triphosphate - pharmacology; Animals; Blood Platelets - drug effects; Blood Platelets - enzymology; Cell Membrane - drug effects; Cell Membrane - enzymology; Drug Synergism; Enzyme Activation - drug effects; guanine nucleotide-binding protein; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate - analogs & derivatives; Guanosine Triphosphate - pharmacology; Inositol Phosphates - blood; Phosphatidylinositols - blood; phospholipase C; platelets; Rabbits; Thionucleotides - pharmacology; thrombin; Thrombin - pharmacology; Type C Phospholipases - blood
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2430457

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5'-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.