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. 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. 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. 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. 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. 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. It was concluded that thrombin-induced hydrolysis of phosphoinositides has characteristics consistent with mediation by a receptor that is similar to that that triggers dense-granule secretion, requires continuous presence of active thrombin to be maintained, is mediated by a receptor that displays thrombin-induced desensitization, and is only partially enhanced by secondary agents.

Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators

“…This would require the continued presence of proteolytically competent thrombin. It is known that hirudin, which rapidly inactivates thrombin and can strip it from its receptors, also quickly terminates PtdOH production (Holmsen et al, 1981;Huang and Detwiler, 1987), AA release and phosphoinositide breakdown (Huang and Detwiler, 1987), and causes accelerated protein differences in desensitization can also be explained by the partial dephosphorylation (this paper). This hypothesis is also supported by Huang et al (1991) who showed that an active-site inhibitor, which does not inhibit thrombin's binding to the receptor, also arrested PtdOH production.…”

Differential dependence of early and late increases in 1,2-diacylglycerol on the presence of catalytically active alpha-thrombin: evidence for regulation at the level of 1,2-diacylglycerol generation.

“…These data suggest the existence of alternate a-thrombin-stimulated pathways involved in generating early (0-1 h) and late (1)(2)(3)(4) h) increases in 1,2-diacylglycerol. An analogous situation is found in platelets, where a-thrombininduced responses can be divided into two types: those that are hirudin-sensitive (and I I I I therefore require the continual presence of ac-Ct: _E CE G tive ca-thrombin) and those that are hirudin-in-'C XC ' ' sensitive (Holmsen eta!., 1981;Huang and Detwiler, 1987). Because a-thrombin is a serine protease as well as a ligand for its own receptor, n a-thrombin-stimulated DNA differential regulation could be occurring at the um-starvedandwashedasde-level of binding and/or substrate recognition.…”

Differences between platelet phosphoinositide metabolism stimulated by thrombin or SFLLRN are not accounted for by interaction of thrombin with glycoprotein Ib

“…Several groups have demonstrated that terminating occupancy by thrombin of its platelet receptor, using hirudin, also stops inositol phospholipid hydrolysis and formation of phosphatidic acid, although aggregation and secretion are not inhibited [27][28][29]. To ensure that our observations were not due to degradation of SFLLRN, which would effectively terminate receptor occupancy, we used the aminopeptidase inhibitor amastatin to show that the decreased formation of IP 3 at 60 sec in SFLLRNstimulated platelets was not caused by degradation of SFLLRN by ectopeptidase.…”