Receptor-Induced Diacylglycerol Formation in Permeabilized Platelets; Possible Role for a Gtp-Binding Protein

Abstract

Exposure of human platelets to 10 discharges from a 4.5 μF capacitor charged at 3 kV permitted isolation of a stable preparation of permeabilized platelets that, after equilibration with Ca²⁺ buffers (pCa < 6) for 15 min at 0°C, secreted 5-hydroxytryptamine (5-HT) at 25°C. Thrombin enhanced the sensitivity to Ca²⁺ of the secretion of 5-HT by about 10-fold, whereas Arg -vasopressin and the prostaglandin endoperoxide analogue, U-46619, increased sensitivity to Ca²⁺ by 3 to 4-fold. This action of thrombin was associated with stimulation of diacylglycerol formation, a marked increase in phosphorylation of protein P47 and a smaller increase in phosphorylation of the P-light chain of myosin. Thrombin exerted these effects at a [Ca²⁺ _free] of 0.1 μM, suggesting that the receptor-activated breakdown of platelet phosphoinositides to diacylglycerol may not require prior Ca²⁺ mobilization in intact platelets. In both the presence and absence of thrombin, a higher [Ca²⁺ _free] was required for optimal secretion than for maximal phosphorylation of P47 and myosin light-chain, indicating that Ca²⁺ and possibly diacylglycerol have roles in the secretory mechanism additional to activation of the enzymes that phosphorylate these proteins. Stable GTP analogues such as guanosine-5′-0-(3-thiotriphosphate) (GTPγS), and to a lesser extent GTP itself, enhanced the Ca²⁺ sensitivity of the secretion of 5-HT from permeabilized platelets. Moreover, GTP potentiated the stimulatory action of thrombin. These effects of GTPγS and GTP were associated with increased diacylglycerol formation and were inhibited by guanosine-5′-0-(2-thiodiphosphate) (GDPβS) suggesting that a GTP-binding protein may play a role in the receptor-activated breakdown of phosphoinositides. However, as GDPβS did not inhibit the potentiation of secretion caused by thrombin alone, a GTP-independent pathway of platelet activation may also exist.