Effects of guanosine 5′‐[γ‐thio]triphosphate and thrombin on the phosphoinositide metabolism of electropermeabilized human platelets
Authors: CULTY Martine , DAVIDSON Monica M. L. , HASLAM Richard J. 
Published: March 2005

Incubation of human platelets with myo‐[3H]inositol in a low‐glucose Tyrode's solution containing MnCl2 enhanced the labelling of phosphoinositides about sevenfold and greatly facilitated the measurement of [3H]inositol phosphates formed by the activation of phospholipase C. Labelled platelets were permeabilized by high‐voltage electric discharges and equilibrated at 0°C with ATP, Ca2+ buffers and guanine nucleotides, before incubation in the absence or presence of thrombin.

Incubation of these platelets with ATP in the presence or absence of Ca2+ ions led to the conversion of [3H]phosphatidylinositol to [3H]phosphatidylinositol 4‐phosphate and [3H]phosphatidylinositol 4,5‐bisphosphate ([3H]PtdInsP2). At a pCa of 6, addition of 100 μM GTP[γS] both prevented this accumulation of [3H]PtdInsP2 and stimulated its breakdown; the formation of [3H]inositol phosphates was increased ninefold. After 5 min these comprised 70% [3H]inositol monophosphate ([3H]InsP), 28% [3H]inositol bisphosphate ([3H]InsP2) and 2% [3H]inositol trisphosphate ([3H]InsP3). In shorter incubations higher percentages of [3H]InsP2 and [3H]InsP3 were found. In the absence of added Ca2+, the formation of [3H]inositol phosphates was decreased by over 90%.

Incubation of permeabilized platelets with GTP[γS] in the presence of 10 mM Li+ decreased the accumulation of [3H]InsP and increased that of [3H]InsP2, without affecting [3H]InsP3 levels. Addition of unlabelled InsP3 decreased the intracellular hydrolysis of exogenous [32P]InsP3 but did not trap additional [3H]InsP3. These results and the time course of [3H]inositol phosphate formation suggest that GTP[γS] stimulated the action of phospholipase C on a pool of [3H]phosphatidylinositol 4‐phosphate that was otherwise converted to [3H]PtdInsP2 and that much less hydrolysis of [3H]phosphatidylinositol to [3H]InsP or of [3H]PtdInsP2 to [3H]InsP3 occurred.

At a pCa of 6, addition of thrombin (2 units/ml)to permeabilized platelets caused small increases in the formation of [3H]InsP and [3H]InsP2. This action of thrombin was enhanced twofold by 10–100 μM GTP and much more potently by 4–40 μM GTP[γS]. In the presence of the latter, thrombin also increased [3H]InsP3. The total formation of [3H]inositol phosphates by permeabilized platelets incubated with thrombin and GTP[γS] was comparable with that observed on addition of thrombin alone to intact platelets. However, HPLC of the [3H]inositol phosphates formed indicated that about 75% of the [3H]InsP accumulating in permeabilized platelets was the 4‐phosphate, whereas in intact platelets stimulated by thrombin, up to 80% was the 1‐phosphate. With both preparations, the [3H]InsP2 formed coeluted with the 1,4‐bisphosphate and [3H]InsP3 largely with the 1,3,4‐trisphosphate. The results provide additional evidence that the activation of phospholipase C by thrombin in the platelet is mediated by a guanine‐nucleotide‐binding protein and indicate that, though PtdIns may be hydrolysed in intact platelets, it is not the major substrate of phospholipase C in permeabilized platelets at a pCa of 6. The formation of the 1,3,4‐trisphosphate isomer of [3H]InsP3 in platelets suggests the presence of an InsP4 pathway.