Michael F. Crouch

Epinephrine induces association of pp60src with Giα in human platelets

Using specific antibodies against the alpha subunit of the inhibitory GTP-binding protein Gi, we analyzed the association of Gi alpha with other cellular components in human platelets. Three tyrosine phosphorylated proteins with molecular mass of 63, 58, and 55 kDa were specifically associated with Gi alpha in resting platelets. Stimulation of platelets with epinephrine, but not with thrombin, induced an increase of the reactivity of the 63- and 55-kDa proteins to anti-phosphotyrosine antibodies on western blotting. By in vitro kinase assay we found that epinephrine induced the association of kinase activity with Gi alpha and that the 63-kDa protein was phosphorylated by this activity. The association of kinase activity with Gi alpha in epinephrine-stimulated platelets paralleled the association of pp60src with Gi alpha, as detected by western blotting analysis using specific anti-pp60src monoclonal antibodies. The interaction of pp60src with Gi alpha may play a role in the mechanism of platelet activation by epinephrine or in the epinephrine-induced potentiation of the action of other platelet agonists.

No direct correlation between Ca2+ mobilization and dissociation of Gi during platelet phospholipase A2 activation

Stimulation of human platelets with thrombin is accompanied by activation of both phospholipases C and A2. These have been considered to be sequential events, with phospholipase A2 activation resulting from the prior hydrolysis of inositol phospholipids and mobilization of intracellular Ca2+ stores. However, our and other laboratories have recently questioned this proposal, and we now present further evidence that these enzymes may be activated by separate mechanisms during thrombin stimulation. Alpha-thrombin induced the rapid hydrolysis of inositol phospholipids, and formation of inositol trisphosphate and phosphatidic acid. This was paralleled by mobilization of Ca2+ from internal stores. These responses were blocked by about 50% by prostacyclin. In contrast, the liberation of arachidonic acid induced by alpha-thrombin was totally inhibited by prostacyclin. The less-effective agonists, platelet activating factor (PAF) and gamma-thrombin also both stimulated phospholipase C, but whereas PAF evoked a rapid and transient response, that of gamma-thrombin was delayed and more sustained. The abilities of these agonists to induce the release of Ca2+ stores closely paralleled phospholipase C activation. However, the maximal intracellular Ca2+ concentrations achieved by these two agents were the same. Despite this, gamma-thrombin and not PAF, was able to release a small amount of arachidonic acid. When alpha-thrombin stimulation of platelets was preceded by epinephrine, there was a potentiation of phospholipase C activation, Ca2+ mobilization and aggregation. The same was true for gamma-thrombin and PAF. However, unlike alpha-thrombin, the gamma-thrombin-stimulated arachidonic acid release was not potentiated by epinephrine, but rather somewhat reduced. These results suggested that phospholipase C and phospholipase A2 were separable events in activated platelets. The mechanism by which alpha-thrombin stimulated phospholipase A2 did not appear to be through dissociation of the inhibitory GTP-binding protein, Gi, since gamma-thrombin decreased the pertussis toxin-induced ADP-ribosylation of the 41 kDa protein as much as did alpha-thrombin, but was a much less effective agent than alpha-thrombin at inducing arachidonic acid liberation.

Phosphorylation of the 47 KDa protein in gamma-thrombin-stimulated human platelets does not activate phospholipase A2: Evidence against lipocortin

Intact human platelets were stimulated with alpha or gamma thrombin in the presence and absence of epinephrine and the ability of these agonists to stimulate aggregation, arachidonic acid release and protein phosphorylation was measured. Epinephrine alone had no effect on any of these events. Both alpha and gamma thrombin induced platelet aggregation which was potentiated in each case by epinephrine. Similarly, both thrombin species were able to induce the phosphorylation of platelet 20 KDa and 47KDa proteins. The gamma thrombin-induced phosphorylation was slightly enhanced by epinephrine. In contrast, only alpha thrombin was capable of inducing significant arachidonic acid release and the small release induced by gamma thrombin was reduced by epinephrine. These results show that the agonist-induced phosphorylation of the 47KDa protein by protein kinase C does not impart the ability to activate phospholipase A2 in human platelets, and questions the suggestion that the 47KDa protein is lipocortin.

Nitric oxide induces polarization of actin in encephalitogenic T cells and inhibits their in vitro trans-endothelial migration in a p70S6 kinase-independent manner

Nitric oxide (NO) inhibits both actively induced and transferred autoimmune encephalomyelitis. To explore potential mechanisms, we examined the ability of NO to inhibit migration of T lymphoblasts through both collagen matrices and monolayers of rat brain endothelial cells. The NO donor 1‐hydroxy‐2‐oxo‐3, 3‐bis (2‐aminoethyl)‐1‐triazene (HOBAT) inhibited migration in a concentration‐dependent manner. NO pretreatment of T cells inhibited migration through untreated endothelial cells, but NO pretreatment of endothelial cells had no inhibitory effect on untreated T cells. Therefore NOs migration inhibitory action was mediated through its effect on T cells and not endothelial cells. HOBAT did not inhibit migration by inducing T‐cell death but rather by polarizing the T cells, resulting in a morphology suggestive of migrating cells. P70S6 kinase, shown to have a role in NO‐induced migration inhibition in fibroblasts, had no role in the inhibitory effect of NO on T‐cell migration. Thus, HOBAT did not alter p70S6K activity nor did rapamycin, a specific inhibitor of p70S6K, inhibit HOBAT‐induced T‐cell morphological changes or T‐cell migration. We suggest that NO‐induced morphological changes result in T cells with predefined migratory directionality, thus limiting the ability of these cells to respond to other migratory signals.

The Na+H+ antiporter is not involved in potentiation of thrombin-induced responses by epinephrine

Stimulation of platelets with thrombin, ADP and epinephrine has recently been shown to activate a Na+/H+ antiporter, with a resulting alkalinization of the cytoplasm. Unlike thrombin, however, epinephrine is incapable of directly activating phospholipase C, but is well known to potentiate the effects of thrombin on this enzyme and other subsequent steps of platelet activation. Therefore, we have studied the involvement of the Na+/H+ antiporter in this aspect of epinephrine action to see whether alkalinization of platelet cytosol could be a requirement for agonists to stimulate inositol phospholipid hydrolysis and mobilize cellular Ca2+ stores. Alpha-thrombin induced the rapid formation of inositol trisphosphate with a parallel mobilization of intracellular Ca2+ stores. Epinephrine alone had no effect on either of these parameters. The response to thrombin desensitized over a period of minutes, and after this had occurred, epinephrine was able to activate phospholipase C and induce the release of intracellular Ca2+. This showed that epinephrine was able to recouple thrombin receptors to phospholipase C, and this appeared to be mediated by the same mechanism which is involved in potentiation by epinephrine of thrombin-stimulation of phospholipase C. These effects of epinephrine were not altered by inhibition of the Na+/H+ antiporter with ethylisopropylamiloride or by use of the Na+/H+ ionophore, monensin. We conclude that epinephrine potentiates thrombin-induced responses by a mechanism which is unrelated to its effects on the Na+/H+ antiporter, and this is not a requirement for thrombin-induced phospholipase C activation.

The Relationship between Phospholipases A2 and C in Signal Transduction

Arachidonic acid can be released from membrane phospholipids of platelets in response to a number of receptor-mediated signals. The enzymes most responsible for this activation are phospholipase A2’ and, to a lesser degree, 1,2-diacylgIycerol lipase.’ In this paper, we will describe some of the studies on phospholipase A2 activation that we have carried out and also some other published work that has helped us understand some of the physiological control mechanisms of this enzyme. The importance of phospholipase A, in receptor-mediated platelet activation varies with both the type and the strength of agonist used. As examples, collagen and epinephrine are absolutely dependent on the release of arachidonic acid for stimulation of platelet secretion and aggregation, whereas thrombin depends on arachidonic acid release only when used at low concentration; at higher doses the ability of thrombin to activate platelets is independent of arachidonic acid metabolites. The most relevant arachidonic acid metabolites for platelet stimulation are endoperoxides and thromboxane A’.

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.

Spacial isolation of protein kinase C activation in thrombin stimulated human platelets.

Thrombin stimulation of human platelets is associated with turnover of inositol phospholipids, mobilization of intracellular Ca2+ stores, and activation of protein kinase C. However, within 5 minutes, the thrombin receptor desensitizes, but can be re-coupled to its effectors by stimulation of alpha 2-adrenergic receptors (Crouch and Lapetina, J. Biol. Chem. 263, 3363-3371, 1988). This effect of epinephrine was found to be inhibited by preincubation of platelets with phorbol ester, suggesting that protein kinase C was inhibitory. However, since thrombin also activated protein kinase C and epinephrine was active following thrombin stimulation of platelets, this implied that thrombin activation of protein kinase C may have been spacially isolated near the thrombin receptor and could not inactivate alpha 2-receptor activity. In the present paper, we have tested this possibility, and we present evidence which strongly favours the possibility that protein kinase C activation by receptors induces its local translocation to the cell membrane.

Multiple Messenger Roles for the Inhibitory GTP-Binding Protein, G i , in Human Platelets

The cellular events which are necessary for transducing signals from the binding of an agonist with its surface membrane receptor to the final cellular response are still much of a mystery. One particular class of receptors stimulates cells by inducing a rise in the cytosolic Ca + concentration and also by activating protein kinase C. To achieve this, the agonist-occupied receptor stimulates phospholipase C which hydrolyses inositol phospholipids, particularly phosphatidylinositol-4,5-bisphosphate. The result is the generation of the second messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, which release intracellular Ca + stores and activate protein kinase C., respectively (Berridge, 1987).