Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

Homeostatic intracellular-free Ca2+ is permissive for Rap1-mediated constitutive activation of alpha4 integrins on eosinophils

Although much progress has been made in understanding the molecular mechanisms underlying agonist-induced "inside-out" activation of integrins, little is known about how basal levels of integrin function are maintained. This is particularly important for nonactivated eosinophils, where intermediate activation of alpha(4)beta(1) integrin supports recruitment to endothelial cells under flow conditions. Depletion of intracellular Ca(2+) and pharmacological inhibition of phospholipase C (but not other intracellular signaling molecules, including PI3K, ERK1/2, p38 MAPK, and tyrosine kinase activity) abrogated basal alpha(4) integrin activity in nonactivated eosinophils. Basal alpha(4) integrin activation was associated with activation of the small GTPase Rap1, a known regulator of agonist-induced integrin function. Basal Rap activation was dependent upon phospholipase C, but not intracellular Ca(2+). However, depletion of intracellular Ca(2+) in CD34(+) hematopoietic progenitor cells abolished RapV12-mediated induction of alpha(4) integrin activity. Thus, residual Rap activity or constitutively active Rap activity in Ca(2+)-depleted cells is not sufficient to induce alpha(4) integrin activation. These data suggest that activation of functional alpha(4) integrin activity in resting eosinophils is mediated by Rap1 provided that the intracellular-free Ca(2+) is at a normal homeostatic concentration.     

Ca2+-mediated activation of phosphofructokinase in perfused rat heart.

Perfusion of the isolated rat heart with Ca2+ concentrations exceeding 3 mM activated phosphofructokinase and phosphorylase, and decreased the concentration of cyclic AMP. Half-maximal activation of phosphofructokinase occurred at 5 mM-CaCl2; significant activation of phosphorylase did not occur until the concentration of CaCl2 exceeded 12 mM. The time course for the activation of phosphofructokinase at 12 mM-CaCl2 indicated that maximal activation occurred within 2 min; when the perfusion-medium Ca2+ concentration was re-adjusted to 3 mM, the phosphofructokinase activity returned to pre-activation values within 30 s. The addition of Ca2+ to extracts of heart did not activate phosphofructokinase. The activation of phosphofructokinase by sub-maximal doses of adrenaline and Ca2+ were not additive. The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol and by 1 microM-isoprenaline was inhibited by high concentrations of K+ (22-56 mM). The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol, 12 mM-CaCl2 and by 1 microM-isoprenaline was blocked by the slow Ca2+-channel blocker nifedipine. These findings suggest that both the beta- and alpha-adrenergic mechanisms for the activation of rat heart phosphofructokinase involve an increase in the myoplasmic Ca2+ concentration. This increase may result from an inhibition of Ca2+ efflux or a stimulation of Ca2+ influx.     

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.     

Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.     

Mathematical model of PAR1-mediated activation of human platelets

Thrombin, one of the major proteases in the coagulation cascade, activates protease activated receptors 1 and 4 (PAR 1 and PAR4) to generate a network of intracellular signals that lead to stable platelet aggregation. Abnormal platelet activation could lead to either thrombosis or bleeding disorders, thus a predictive model of platelet activation would be an invaluable tool for the study of platelet function. In this work, we developed a computational model of PAR1-stimulated human platelet activation fully based on experimental observations. The model is represented by a system of ordinary differential equations (ODEs) describing the kinetics of the interacting components. The model is able to reproduce experimental dose responses and time-courses of cytosolic calcium (Ca(2+)), phosphatidylinositol 4,5-bisphosphate (PIP2), diacylglycerol (DAG), GTP-bound Ras-proximate-1 (Rap1GTP), secretion of dense-granules, and activation of integrin α2bβ3 (GPIIbIIIa). Because of the inherent complexity of such a model, we also provide a simple way to identify and divide the system into interlinked functional modules to reduce the number of unknown parameters. Both the full and the reduced kinetic models are shown to predict platelet behavior in response to PAR1 activation.     

Ca2+-mediated association of glycoprotein G (thrombinsensitive protein, thrombospondin) with human platelets

Washed human platelets suspended in buffers containing either 1.8 mM Ca2+ and 0.49 mM Mg2+ or 1 mM EDTA were treated with human alpha-thrombin to induce secretion. Glycoprotein G, a major glycoprotein in alpha-granules, was quantitatively secreted from platelets activated in the EDTA-containing buffer but remained with the platelet in the presence of Ca2+ and Mg2+. Addition of Ca2+ to the platelets that were activated in the presence of EDTA caused glycoprotein G to bind to platelets. To determine if glycoprotein G is expressed on the membrane surface of the activated platelet, platelets were rapidly labeled by a method employing lactoperoxidase-catalyzed iodination. Although glycoprotein G was barely detected on the surface of unstimulated platelets, labveling 1 min after thrombin treatment showed that glycoprotein G rapidly became one of the prominent surface proteins. These findings show that an alpha-granule protein, glycoprotein G, is one of the major glycoproteins on the membrane surface of thrombin-activated platelets and that its binding is dependent on divalent cations.     

Dihydropyridine-sensitive and insensitive Ca2+ channels in human platelets

The Ca2+ channel blocker, nifedipine, a dihydropyridine derivative, inhibited the Ca2+ influx and release from internal stores caused by collagen or a low concentration of the thromboxane A2 (TXA2) analogue, 9,11-epithio-11,12-methano-TXA2 (STA2) (10 nM), but did not inhibit those caused by thrombin or a high concentration of STA2 (100 nM). These results indicate the presence of two distinct, dihydropyridine-sensitive and insensitive, Ca2+ channels dependent on the concentrations and classes of agonists in human platelets.     

Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation

Like other small G proteins of the Ras superfamily, Rap1 is activated by distinct guanine nucleotide exchange factors (GEFs) in response to different signals to elicit cellular responses. Activation of Rap1 by cyclic AMP (cAMP) can occur via cAMP-dependent protein kinase A (PKA)-independent and PKA-dependent mechanisms. PKA-independent activation of Rap1 by cAMP is mediated by direct binding of cAMP to Rap1-guanine nucleotide exchange factors (Rap1-GEFs) Epac1 (exchange protein directly activated by cAMP 1) and Epac2 (Epac1 and Epac2 are also called cAMP-GEFI and -GEFII). The availability of cAMP analogues that selectively activate Epacs, but not PKA, provides a specific tool to activate Rap1. It has been argued that the inability of these analogues to regulate extracellular signal-regulated kinases (ERKs) signaling despite activating Rap1 provides evidence that Rap1 is incapable of regulating ERKs. We confirm that the PKA-independent activation of Rap1 by Epac1 activates a perinuclear pool of Rap1 and that this does not result in ERK activation. However, we demonstrate that this inability to regulate ERKs is not a property of Rap1 but is rather a property of Epacs themselves. The addition of a membrane-targeting motif to Epac1 (Epac-CAAX) relocalizes Epac1 from its normal perinuclear locale to the plasma membrane. In this new locale it is capable of activating ERKs in a Rap1- and cAMP-dependent manner. Rap1 activation by Epac-CAAX, but not wild-type Epac, triggers its association with B-Raf. Therefore, we propose that its intracellular localization prevents Epac1 from activating ERKs. C3G (Crk SH3 domain Guanine nucleotide exchanger) is a Rap1 exchanger that is targeted to the plasma membrane upon activation. We show that C3G can be localized to the plasma membrane by cAMP/PKA, as can Rap1 when activated by cAMP/PKA. Using a small interfering RNA approach, we demonstrate that C3G is required for the activation of ERKs and Rap1 by cAMP/PKA. This activation requires the GTP-dependent association of Rap1 with B-Raf. These data demonstrate that B-Raf is a physiological target of Rap1, but its utilization as a Rap1 effector is GEF specific. We propose a model that specific GEFs activate distinct pools of Rap1 that are differentially coupled to downstream effectors.     

Cyclic AMP stimulates Ca(2+)-ATPase-mediated Ca2+ extrusion from human platelets.

The effect of cAMP on active Ca2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca2+ in the cytoplasmic compartment ([Ca2+]cyt). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca2+]cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147-163). Forskolin, at a maximally effective concentration of 10 microM, was shown to stimulate Ca2+ extrusion by increasing by a factor of 1.6 +/- 0.5 the Vm of a saturable component, previously identified with a Ca(2+)-Mg(2+)-ATPase located in the plasma membrane. Neither the Km (80 nM) or Hill coefficient (1.7 +/- 0.3) of the Ca(2+)-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na+/Ca2+ exchanger) over the [Ca2+]cyt range examined (50-1500 nM). Dibutyryl-cAMP (Bt2-cAMP, 1 mM) stimulated the Ca(2+)-Mg(2+)-ATPase component of Ca2+ extrusion by a factor of 2.0 +/- 0.6. Separate experiments showed that 10 microM forskolin reduces the resting [Ca2+]cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in Vm of the pump, countered by a 37-74% increase in the rate constant for passive Ca2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca2+]cyt and (b) increasing the rate of Ca2+ extrusion after the initial influx or triggered release event.     

ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.

Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.     

Stimulation of dense tubular Ca2+ uptake in human platelets by cAMP.

Elevation of intracellular cAMP is shown to increase the rate (V) and maximal extent of Ca2+ uptake by the dense tubules in intact human platelets. Elevation of [cAMP] was accomplished by preincubation with the adenylate cyclase activator forskolin or with dibutyryl-cAMP (Bt2-cAMP). The free concentration of Ca2+ in the dense tubular lumen ([Ca2+]dt) was monitored using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. The free cytoplasmic Ca2+ concentration ([Ca2+]cyt) was monitored in parallel experiments with quin2. Both [Ca2+]cyt and [Ca2+]dt were analyzed in terms of competition between pump and leak mechanisms in the plasma membrane (PM) and dense tubular membrane (DT). When platelets are incubated in media with approx. 1 microM external Ca2+, [Ca2+]cyt is approx. 50 nM and [Ca2+]dt is very low. When 2 mM external Ca2+ is added, [Ca2+]cyt rises to approx. 100 nM and the process of dense tubular Ca2+ uptake can be resolved. Forskolin (10 microM) and Bt2-cAMP increase the rate of dense tubular Ca2+ uptake (V) to 2.1 +/- 0.60 and 1.70 +/- 40 times control values (respectively). The agents also increase the final [Ca2+]dt to 1.70 +/- 0.21 and 1.72 +/- 0.60 times control values (respectively). Titrations with ionomycin (Iono) showed that the increase was due to an increase in the Vm of the dense tubular Ca2+ pump. With [Iono] = 500 nM, [Ca2+]cyt was raised to greater than or equal to 1.0 microM and Vm of the dense tubular pump was elicited. (At [Iono] = 1.0 microM, the final [Ca2+]dt values were degraded 15% due to shunting of Ca2+ uptake.) Analysis showed that forskolin (10 microM) and Bt2-cAMP (1 mM) increase the Vm by a factors of 1.56 +/- 40 and 1.56 +/- 40, respectively. Analysis showed that neither agent changed the Km of the pump significantly from its control value of 180 nM. Neither agent changed the rate constant for passive leakage of Ca2+ across the DT membrane (1.7 min-1).     

Discrete intracellular Ca2+ pools coupled to two distinct Ca2+ influx pathways in human platelets

Ca(2+) influx is an important event associated with platelet activation and regulated by the content of intracellular Ca(2+). Previous studies have suggested two different Ca(2+) pools and two Ca(2+) influx pathways exist in platelets. In the present study, we have investigated the regulation of thrombin- and thapsigargin-induced Ca(2+) entry into human platelets, using fluorescent indicators to monitor Ca(2+) mobilization and membrane potential. It was found that depletion of thapsigargin-sensitive Ca(2+) stores was coupled to Ca(2+) influx through a Ca(2+)-selective pathway. Additional release of Ca(2+) from the thapsigargin-insensitive pool by thrombin caused the opening of a nonselective cation channel.     

Elevated cytosolic Ca2+ activates phospholipase D in human platelets

We have examined the activation of phospholipase D in human platelets treated with alpha-thrombin. When incubated with 1-O-[9,10-3H2]hexadecyl-2-lysophosphatidylcholine (PtdCho) and 1-alkyl-[32P]lysoPtdCho for 2 h, platelets formed 3H/32P-labeled PtdCho in a ratio of 11:1. After incubation of such labeled platelets with alpha-thrombin for 5 min, increased accumulation of 3H/32P-labeled phosphatidic acid (PtdOH) was detected in the same ratio, indicating the action of phospholipase D. The Ca2+ ionophore A23187 and alpha-thrombin each stimulated the formation of labeled PtdOH as above in a time- and concentration-dependent manner, with only minor changes in labeled diglyceride. A23187 was able to cause increases in labeled PtdOH comparable to those observed with alpha-thrombin. beta-Phorbol 12,13-dibutyrate, an activator of protein kinase C, only slightly stimulated the accumulation of labeled PtOH. The protein kinase C inhibitor, staurosporine, totally blocked these changes but only slightly inhibited the increases in labeled PtdOH promoted by alpha-thrombin. These results suggest that an increase in intracellular Ca2+, rather than protein kinase C activity, is a major factor regulating phospholipase D in platelets exposed to alpha-thrombin. We have also examined the relative contributions of phospholipase D and diglyceride kinase (following phospholipase C action) to PtdOH accumulation in [32P]Pi-labeled platelets by comparing the 32P-specific radioactivities of PtdOH, PtdCho, and metabolic gamma-ATP in control and alpha-thrombin-exposed platelets. Based on these determinations, we conclude that 13 and 87% of incremental PtdOH in human platelets exposed to alpha-thrombin arises via phospholipase D acting on PtdCho and phospholipase C/diglyceride kinase, respectively.     

Ca2+ homeostasis in unstimulated platelets

Unstimulated platelets maintain a low cytosolic free Ca2+ concentration and a steep plasma membrane Ca2+ gradient. The mechanisms that are required have not been completely defined. In the present studies, 45Ca2+ was used to examine the kinetics of Ca2+ exchange in intact unstimulated platelets. Quin2 was used to measure the cytosolic free Ca2+ concentration. Under steady-state conditions, the maximum rate of Ca2+ exchange across the platelet plasma membrane, 2 pmol/10(8) platelets/min, was observed at extracellular free Ca2+ concentrations 20-fold less than in plasma. Two intracellular exchangeable Ca2+ pools were identified. The size of the more rapidly exchanging pool (t 1/2, 17 min) and the cytosolic free Ca2+ concentration were relatively unaffected by large changes in the extracellular Ca2+ concentration. In contrast, the size of the more slowly exchanging Ca2+ pool (t 1/2, 300 min) varied with the extracellular Ca2+ concentration, which suggests that it is physically as well as kinetically distinct from the rapidly exchangeable Ca2+ pool. The locations of the Ca2+ pools were determined by differential permeabilization of 45Ca2+-loaded platelets with digitonin. 45Ca2+ in the rapidly exchanging pool was released with lactate dehydrogenase, which suggests that it is located in the cytosol. 45Ca2+ in the slowly exchanging pool was released with markers for both the dense tubular system and mitochondria, but inhibition of mitochondrial Ca2+ uptake with carbonyl cyanide m-chlorophenylhydrazone had no effect on the size of the slowly exchangeable Ca2+ pool or the cytosolic free Ca2+ concentration. In contrast, addition of metabolic inhibitors (KCN plus carbonyl cyanide m-chlorophenylhydrazone plus deoxyglucose) or trifluoperazine caused a decrease in the size of the slowly exchangeable Ca2+ pool and an increase in the cytosolic free Ca2+ concentration. These observations suggest that Ca2+ homeostasis in unstimulated platelets is maintained by limiting Ca2+ influx from plasma, actively promoting Ca2+ efflux, and sequestering Ca2+ within an internal site, which is most likely the dense tubular system and not mitochondria.     

Effects of the antithrombitic agent PCA 4230 on agonist-induced Ca2+ entry and Ca2+ release in human platelets.

We have studied the effects of the antithrombitic agent PCA 4230 on the entry of Mn2+, used here as a Ca2+ surrogate for Ca2+ channels, and on the release of Ca2+ from the intracellular stores in stimulated human platelets loaded with fura-2. PCA 4230 prevented receptor-operated calcium entry activated by thrombin, ADP and collagen with no modification of the Ca2+ release from the intracellular stores. PCA 4230 also inhibited cytochrome P-450-mediated O-dealkylase activity with the same concentration-dependence as the thrombin-induced Mn2+ entry. These results suggest that the inhibitory effects of PCA 4230 on Ca2+ influx may be due to its interaction with cytochrome P-450, which has been proposed recently to be involved in the activation of receptor-operated Ca2+ channels. In addition, PCA 4230 inhibited both PAF-induced Ca2+ entry and Ca2+ release, behaving as a PAF-antagonist. All these effects contribute to explain the antithrombitic action of PCA 4230.     

The possible involvement of protein phosphatase 1 in thrombin-induced Ca2+ influx of human platelets

Protein phosphatase 1 is considered to be involved in thrombin-induced platelet activation (Murata et al., Biochem Int 26:327–334, 1992). To clarify the mechanism, we examined the effects of protein phosphatase 1 and 2A inhibitors (calyculin A, tautomycin, okadaic acid) on Ca²⁺ influx. In the presence of 1 mM Ca²⁺, thrombin- (0.1 U/ml) induced platelet aggregation and ATP release were inhibited by calyculin A, while this inhibitory effect was abolished in the absence of Ca²⁺ (EGTA 1 mM). Furthermore, thrombin-induced Mn²⁺ influx but not intracellular Ca²⁺ mobilization was inhibited by calyculin A in a dose-related manner. Calyculin A also blocked the ongoing Ca²⁺ influx when added 3 min after thrombin stimulation. Similar inhibitory effects were observed with okadaic acid and tautomycin in the same potency sequence as the reported one for protein phosphatase 1 (calyculin A > tautomycin > okadaic acid). These results suggest that the anti-platelet effects of phosphatase inhibitors are due to the inhibition of Ca²⁺ influx and that protein phosphatase 1 plays a key role in the regulation of receptor operated Ca²⁺ channel of human platelets.     

A Gi-dependent pathway is required for activation of the small GTPase Rap1B in human platelets

Stimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcgammaRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca(2+) concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcgammaRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the G(i)-coupled ADP receptor P2Y12, but was not affected by blockade of the G(q)-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca(2+) mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca(2+) signaling. In platelets stimulated by cross-linking of FcgammaRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the G(i)-coupled alpha(2A)-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a G(q)-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the G(q)-coupled thromboxane A(2) receptor by was completely inhibited by ADP scavengers under conditions in which intracellular Ca(2+) mobilization was unaffected. Inhibition of -induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a G(i)-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B.