Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization

Abstract

The relationship between occupancy of thrombin receptors on platelets and enhanced phosphoinositide hydrolysis was analysed by examination of the dose-response relationship, the effects of thrombin inhibitors and the contribution of secondary effects. Washed human platelets were labelled with [3H]inositol, and agonist-induced accumulation of labelled inositol phosphates was measured. (1) The dose-response curves and the time courses for .alpha.-thrombin- or .gamma.-thrombin-induced accumulation of inositol phosphates were similar to those for dense-granule secretion. (2) Addition of the thrombin inhibitor hirudin to thrombin-activated platelets revealed that the continuous presence of active thrombin was required to maintain the accumulation of labelled inositol phosphates; the total production of inositol phosphates increased with longer periods of exposure to thrombin, reaching a maximum between 5 and 10 min. (3) After activation with thrombin, the ability of a second, greater, addition of thrombin to induce additional phosphoinositide hydrolysis decreased with time; it was absent within 10 min after the first addition. (4) The failure to sustain accumulation of labelled inositol phosphates or to respond to a second addition of thrombin beyond 10 min was not due to depletion of the pool of labelled precursors, because the platelets retained their ability to respond to collagen. (5) Addition of ADP-consuming enzymes decreased sensitivity to thrombin, but inhibition of cyclo-oxygenase with indomethacin did not impair the thrombin-induced hydrolysis of phosphoinositides. (6) It was concluded that thrombin-induced hydrolysis of phosphoinositides (i) has characteristics consistent with mediation by a receptor that is similar to that that triggers dense-granule secretion, (ii) requires continuous presence of active thrombin to be maintained, (iii) is mediated by a receptor that displays thrombin-induced desensitization, and (iv) is only partially enhanced by secondary agents.