The Effects of Thrombin on Adenyl Cyclase Activity and a Membrane Protein from Human Platelets

Abstract

A B S T R A C T Washed human platelets were incubated with 0.1-1.0 U/ml human thrombin and the effects on adenyl cyclase activity and on a platelet membrane protein (designated thrombin-sensitive protein) were studied. Adenyl cyclase activity was decreased 70-90% when intact platelets were incubated with thrombin. The Ti for loss of adenyl cyclase activity was less than 15 sec at 1 U/ml thrombin. There was no decrease of adenyl cyclase activity when sonicated platelets or isolated membranes were incubated with these concentrations of thrombin. Loss of adenyl cyclase activity was relatively specific since the activities of other platelet membrane enzymes were unaffected by thrombin. Prior incubation of platelets with dibutyryl cyclic adenosine monophosphate (AMP), prostaglandin El, or theophylline protected adenyl cyclase from inhibition by thrombin.Incubation of intact but not disrupted platelets with thrombin resulted in the release of thrombin-sensitive protein from the platelet membrane. The rapid release of this protein (Ti < 15 sec) at low concentrations of thrombin suggested that removal of thrombin-sensitive protein from the platelet membrane is an integral part of the platelet release reaction. This hypothesis is supported by the parallel effects of thrombin on adenyl cyclase activity and thrombin-sensitive protein release in the presence of dibutyryl cyclic AMP, prostaglandin Ei, and theophylline at varying concentrations of thrombin.

Thrombin-induced protein phosphorylation in human platelets.

“…The thrombin concentration curve and the time-course for thrombin-induced phosphorylation of peak 7 and peak 9 proteins were similar to those parameters previously observed for the thrombin-induced platelet release reaction and for the inhibition of platelet adenylate cyclase by thrombin (5,33,38,39).…”

“…The published time of 10-30 s for half-maximal thrombin-induced release (33,38,39) is similar to the observed time for halfmaximal thrombin-induced phosphorylation of protein peaks 7 and 9 (10-14 s). Likewise, the concentration of thrombin required for half-maximal release (5,38,39) is similar to the observed concentration of thrombin (0.25 U/ml) required for half-maximal phosphorylation of peak 7 and peak 9 proteins. DFP-treated thrombin, which binds normally to platelets without inducing the release reaction (3), does not induce peak 7 protein or peak 9 protein phosphorylation.…”

“…The lectin E-PHA, which binds to the platelet surface, decreases adenylate cyclase activity, and causes the platelet release reaction in washed human platelets (14-33), induces peak 7 protein and peak 9 protein phosphorylation. Thrombin and E-PHA are presumably related in their mode of action on platelets since elevation of intracellular platelet cAMIP blocks their initiation of the release reaction (5,33,40). We observed that PGE1 or DB-cAMP inhibited thrombin-induced phosphorylation of peak 7 and 9 proteins.…”

“…Thrombin, a potent inducer of the platelet release reaction, has been shown to bind to receptors on the platelet surface (3,4) and to decrease the activity of platelet adenylate cyclase without a change in phosphodiesterase activity (5). In other tissues the effects of cAMP are mediated through changes in protein phosphorylation resulting from a cAMP-induced alteration in protein kinase and/ or phosphoprotein phosphatase activity (648).…”

The perturbation of thrombin binding to human platelets by anions.

“…Two types of binding were observed with thrombin of both species; high affinity binding (KdI s-0.02 U/ml) of a small amount of thrombin (a 500 molecules/platelet) and a larger amount of thrombin binding of lower affinity (Kdiss -2 U/ml) with 30,000-50,000 thrombin molecules binding per platelet at saturation. The amount of thrombin required to induce 50% ["C] serotonin release in this system is about 0.03-0.1 U/ml while the amount of thrombin required for 50% inhibition of adenylate cyclase activity or release of the thrombin-sensitive protein (2,3) or for maximal stimulation of protein phosphorylation (4) is about 0.2-0.4 U/ml. Thus, full saturation of thrombin receptor sites occurs at concentrations 10-fold higher than required to elicit the physiological response of platelets.…”