Regulation of hormone-induced Ca2+ mobilization in the human platelets.

alpha-Thrombin, gamma-thrombin, and platelet-activating factor each stimulated the mobilization of intracellular Ca2+ stores in aspirin-treated human platelets. This was followed by desensitization of the receptors, as shown by the return of the Ca2+ level to basal values and by the fact that a subsequent addition of a second different agonist, but not the same agonist, could again elicit a response. Epinephrine, acting on alpha 2-adrenergic receptors, was by itself ineffective at mobilizing Ca2+ stores. However, when added after the thrombin-induced response, epinephrine could evoke a considerable release of Ca2+ from cellular stores. This appeared to be due to epinephrine recoupling thrombin receptors to phospholipase C. In support of this, epinephrine was able to induce the formation of inositol triphosphate when added after the response to thrombin had also become desensitized. Alone, epinephrine was without effect. Pre-activation of protein kinase C with the phorbol ester abolished these effects of epinephrine, suggesting that epinephrine was working by activating a protein which could be inactivated by phosphorylation. Our current work is to characterize this protein that may be a member of the Gi, GTP-binding protein family.


Introduction
In many mammalian cell types, stimulation of specific receptors by agonists is accompanied by a nonvoltage-regulated mobilization of intracellular Ca2" stores. This results in the elevation of the cytosolic Ca2" concentration, which can evoke cellular responses such as in the platelet, secretion, shape change, and aggregation. The Ca2" appears to originate from specific, nonmitochondrial sites in the cell, probably the endoplasmic reticulum or the plateletdense tubular system.
The mechanism by which agonists cause a receptormediated Ca2"-mobilization has been extensively studied over recent years. It appears that the transduction mechanism of occupied receptors is the phospholipase C-catalyzed hydrolysis of inositol phospholipid, specifically, phosphatidylinositol-4,5bisphosphate (PIP2). The immediate products of this reaction are diacyglycerol (DAG), which can activate protein kinase C (1) and inositol trisphosphate (1P3), which will mobilize the hormone-sensitve Ca2+ stores when applied to permeabilized cells (2). Therefore, the connection between receptor and Ca2" store mobilization has been quite convincingly were a) what are the kinetics of Ca2" mobilization in the platelet; b) did these correlate with changes in the IP3 levels; and c) where in the chain of events following the agonist's receptor occupation is the release of Ca2" modulated or controlled? We have discussed these points elsewhere (3).

Platelet Ca2+ Responses to Agonists
We chose three different agonists: a-thrombin, ythrombin, and the platelet-activating factor (PAF) to study Ca2" mobilization in the platelet. Each of these agents was able to mobilize Ca2`from intracellular Ca2+ stores, but to ranging degrees and rates (Fig. 1). a-thrombin was most effective, whereas y-thrombin produced a smaller and slower response. PAF produced a rapid Ca2`signal, but had a smaller magnitude than that of y-thrombin.
Each of these responses was quite transient, despite the continued presence of the agonist; after the return to baseline levels, the cytosolic Ca2`level could not be reelevated by a second addition of the same agonist at the same concentration ( Fig. 1). Thus, the loss of response was not due to degradation of the agonist and could not be explained by the agonists fully depleting the Ca2`stores, since PAF was a much less effective agonist than aor y-thrombin. The Ca2r esponse to PAF returned most quickly to basal levels and could not be restimulated with PAF. These responses appeared to be a desensitization of the receptors or of a post-receptor mechanism. To determine which of these two possibilities was more likely, the ability of a second agonist to elicit the release of Ca2' stores was examined. After the aor ythrombin induced Ca2" release was complete, the cytosolic Ca2" concentration had returned to basal levels, and the addition of the same agonist at the same concentration was shown to have no effect. PAF was still able to induce a small but significant release of Ca2' stores ( Fig. 1). The smaller response to PAF after the thrombin rather than to PAF alone is probably because the thrombin has already depleted much of the Ca2' store, and the PAF can act to release only a much-reduced pool of Ca2".

Thrombin Receptor Desensitization
Because thrombin and PAF are both thought to couple through the same second messenger system (i.e., phospholipase C), this suggests that the desensitization observed is homologous and occurs at the receptor-effector site rather than a post-receptor site. It has been shown previously (4) that activation of protein kinase C, which occurs during stimulation of platelets with thrombin or PAF, is able to increase the degredation of cytosolic IP3 by elevating IP3 phosphatase activity. This could decrease the receptoractivated Ca2" release. However, the activation of protein kinase C does not appear to be the mechanism of the desensitization observed here since this would cause a heterologous desensitization. The desensitization is better explained by being related to the generation of the Ca2'-mobilizing signal (i.e., IP3) rather than to the fate of IP3 once it formed.

Thrombin Effects Are Restored by Epinephrine
To examine this effect further, we have used epinephrine, which is able to potentiate the action of many platelet agonists without having a direct effect on platelet responses. Epinephrine acts via a2-adrenergic receptors, since yohimbine was found to totally inhibit its actions. We found that epinephrine alone had no effect on platelet Ca2' levels. However, when added to platelets previously desensitized to thrombin, epinephrine could elicit a relatively large mobilization of Ca2' stores (Fig. 2). Thus, epinephrine was able to resensitize the thrombin receptor to the generation of a Ca21 signal.

Phospholipase C Activation by Thrombin
In supporting the role of IP3 in mobilizing intracellular Ca2' stores, the desensitization of the thrombininduced Ca2' release and resensitization by epinephrine were paralled by desensitization and resensitization of the a-thrombin-induced IP3 formation (Fig. 3). This showed us that the thrombin receptor did desensitize at the level of receptor activation of phospholipase C and hydrolysis of PIP2, but that the a2-adrenergic receptor could resensitize thrombin receptor coupling. Those effects were able to fully explain our Ca2' data. The next part of our study was to try to assess what caused the homologous desensitizations. Since each desensitization was coupled to specific receptors but showed similar characteristics, it was likely that the agonists induced a local activation of a second messenger, which was itself responsible for the receptor inactivation. It is known from studies of other cell types that protein kinase C activation can be inhibitory to hormone-induced phospholipase C. In the platelet, diacylglycerol, the endogenous protein kinase C activator, is rapidly converted to phosphatidic acid. Thus, diacylglycerol may well be produced as a local event and also locally activate protein kinase C near the occupied receptors.
When we treated platelets with the protein kinase C-activating phorbol ester, phorbol-12,13-dibutyrate (Pdbu), we found a reduction in the thrombin-induced release of Ca2' stores and an abolition of resensitization of thrombin action by epinephrine (Fig. 4). This effect of Pdbu was half maximal at 1 nM and occurred within 30 sec, with both parameters being consistent with a specific activation of protein kinase C. From these results, we suggest that a local elevation in the DAG near the occupied receptor causes a local activation and translocation of protein kinase C so that only the occupied receptor is desensitized.

Conclusion
We believe that receptor desensitization in the human platelet represents an important negative feedback system in controlling platelet responses, including the mobilization of intracellular Ca2" stores and activation of phospholipase C. This desensitization appears to be homologous and mediated by hormone-induced activation of protein kinase C. Our current work (3) implicates the inhibitory GTP-binding protein, Gi, as the substrate of protein kinase C involved in receptor-phospholipase C coupling.