Effects of guanosine 5′‐[γ‐thio]triphosphate and thrombin on the phosphoinositide metabolism of electropermeabilized human platelets

Incubation of human platelets with myo ‐[ 3 H]inositol in a low‐glucose Tyrode's solution containing MnCl 2 enhanced the labelling of phosphoinositides about sevenfold and greatly facilitated the measurement of [ 3 H]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, Ca 2+ 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 Ca 2+ ions led to the conversion of [ 3 H]phosphatidylinositol to [ 3 H]phosphatidylinositol 4‐phosphate and [ 3 H]phosphatidylinositol 4,5‐bisphosphate ([ 3 H]PtdIns P 2 ). At a pCa of 6, addition of 100 μM GTP[γS] both prevented this accumulation of [ 3 H]PtdIns P 2 and stimulated its breakdown; the formation of [ 3 H]inositol phosphates was increased ninefold. After 5 min these comprised 70% [ 3 H]inositol monophosphate ([ 3 H]Ins P ), 28% [ 3 H]inositol bisphosphate ([ 3 H]Ins P 2 ) and 2% [ 3 H]inositol trisphosphate ([ 3 H]Ins P 3 ). In shorter incubations higher percentages of [ 3 H]Ins P 2 and [ 3 H]Ins P 3 were found. In the absence of added Ca 2+ , the formation of [ 3 H]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 [ 3 H]Ins P and increased that of [ 3 H]Ins P 2 , without affecting [ 3 H]Ins P 3 levels. Addition of unlabelled Ins P 3 decreased the intracellular hydrolysis of exogenous [ 32 P]Ins P 3 but did not trap additional [ 3 H]Ins P 3 . These results and the time course of [ 3 H]inositol phosphate formation suggest that GTP[γS] stimulated the action of phospholipase C on a pool of [ 3 H]phosphatidylinositol 4‐phosphate that was otherwise converted to [ 3 H]PtdIns P 2 and that much less hydrolysis of [ 3 H]phosphatidylinositol to [ 3 H]Ins P or of [ 3 H]PtdIns P 2 to [ 3 H]Ins P 3 occurred. At a pCa of 6, addition of thrombin (2 units/ml)to permeabilized platelets caused small increases in the formation of [ 3 H]Ins P and [ 3 H]Ins P 2 . 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 [ 3 H]Ins P 3 . The total formation of [ 3 H]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 [ 3 H]inositol phosphates formed indicated that about 75% of the [ 3 H]Ins P 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 [ 3 H]Ins P 2 formed coeluted with the 1,4‐bisphosphate and [ 3 H]Ins P 3 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 [ 3 H]Ins P 3 in platelets suggests the presence of an Ins P 4 pathway.