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.     

Involvement of SNARE proteins in thrombin-induced platelet aggregation: evidence for the relevance of Ca2+ entry

Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca(2+) mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca(2+) entry, the main mechanism for Ca(2+) influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca(2+) entry for full activation. Depletion of the intracellular Ca(2+) stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca(2+) concentration reached a critical level (around 1.5 microM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca(2+) mobilization in human platelets.     

Regulation of plasma membrane Ca2+-ATPase by small GTPases and phosphoinositides in human platelets

We have investigated the restoration of [Ca(2+)](i) in human platelets following the discharge of the intracellular Ca(2+) stores. We found that the plasma membrane Ca(2+)-ATPase is the main mechanism involved in Ca(2+) extrusion in human platelets. Treatment of platelets with the farnesylcysteine analogs, farnesylthioacetic acid and N-acetyl-S-geranylgeranyl-l-cysteine, inhibitors of activation of Ras proteins, accelerated the rate of decay of [Ca(2+)](i) to basal levels after activation with thapsigargin combined with a low concentration of ionomycin, indicating that Ras proteins are involved in the negative regulation of Ca(2+) extrusion. Rho A, which is involved in actin polymerization, was not responsible for this effect. Consistent with this, the actin polymerization inhibitors, cytochalasin D and latrunculin A, did not alter the recovery of [Ca(2+)](i). Activation of human platelets with thapsigargin and ionomycin stimulated the tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase, a mechanism that was inhibited by farnesylcysteine analogs, suggesting that Ras proteins could regulate Ca(2+) extrusion by mediating tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase. Treatment of platelets with LY294002, a specific inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinase, resulted in a reduction in the rate of recovery of [Ca(2+)](i) to basal levels, suggesting that the products of these kinases are involved in stimulating Ca(2+) extrusion in human platelets.     

Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets

BACKGROUND: Platelet activation leads to the loss of a natural asymmetry of membrane phospholipids (PL) and the subsequent exposure of negatively charged PL in platelets with procoagulant activity that can be monitored routinely with annexin V (AN-V). METHODS: Flow cytometric analysis of merocyanine 540 (MC540) binding may be the alternate choice for the monitoring of platelet procoagulant activity. Due to the increased partition of negatively charged phosphatidylserine (PS) in the membrane outer leaflet of activated platelets, the interaction with MC540 is reduced. RESULTS: Collagen, which facilitated platelet PL bilayer symmetrization, vastly reduced MC540 fluorescence and augmented AN-V binding to platelets. Such a collagen-induced symmetrization was further augmented in the presence of thrombin receptor-activating peptide (TRAP, SFLLRNPNDKYEPF). In the presence of VO(4) ((-3)) (the inhibitor of aminophospholipid translocase), the rebuilt of membrane asymmetry was attenuated, which resulted in further reduced MC540 fluorescence and enhanced AN-V binding in activated cells. In platelets incubated with thapsigargin, the inhibitor of platelet tubular system Ca(2+) ATP-ase, which elevates intraplatelet Ca(2+) concentration, TRAP increased AN-V and reduced MC540 binding. The chelating of Ca(2+) with EGTA outside of activated platelets reduced AN-V binding, but did not affect MC540-positive platelets. The fluctuations in reduced staining with MC540 paralleled enhanced AN-V binding (r = -0.481, P < 0.01), especially for strong "procoagulant" activating agents. CONCLUSIONS: (1) MC540 may be used in whole blood flow cytometry for the monitoring of platelet membrane symmetrization as an alternate or compounding method to AN-V. (2) Platelet staining with MC540 is sensitive to the fluctuations in the intraplatelet [Ca(2+)] during platelet activation. (3) Use of MC540 is characterized by improved diagnostic precision and reliability compared with AN-V.     

Purification and characterization of a calpain activator from human platelets.

A calpain (Ca(2+)-activated neutral protease) activator was purified from human platelets by ammonium sulfate fractionation, gel-filtration, ion-exchange chromatography, followed by heat-treatment. The purified calpain activator with a Mr of 47.5 kDa was a heat-stable protein as demonstrated in other cells. The calpain activator did not change the Ca2+ sensitivity of calpain but activated calpain activity about 2-fold. This calpain activator may play an important role in the activation of the protease system leading to the Ca(2+)-mediated physiological process of platelets.     

Cyclic nucleotides modulate store-mediated calcium entry through the activation of protein-tyrosine phosphatases and altered actin polymerization in human platelets

Agonists elevate the cytosolic calcium concentration in human platelets via a receptor-operated mechanism, involving both Ca(2+) release from intracellular stores and subsequent Ca(2+) entry, which can be inhibited by platelet inhibitors, such as prostaglandin E(1) and nitroprusside which elevate cAMP and cGMP, respectively. In the present study we investigated the mechanisms by which cAMP and cGMP modulate store-mediated Ca(2+) entry. Both prostaglandin E(1) and sodium nitroprusside inhibited thapsigargin-evoked store-mediated Ca(2+) entry and actin polymerization. However, addition of these agents after induction of store-mediated Ca(2+) entry did not affect either Ca(2+) entry or actin polymerization. Furthermore, prostaglandin E(1) and sodium nitroprusside dramatically inhibited the tyrosine phosphorylation induced by depletion of the internal Ca(2+) stores or agonist stimulation without affecting the activation of Ras or the Ras-activated phosphatidylinositol 3-kinase or extracellular signal-related kinase (ERK) pathways. Inhibition of cyclic nucleotide-dependent protein kinases prevented inhibition of agonist-evoked Ca(2+) release but it did not have any effect on the inhibition of Ca(2+) entry or actin polymerization. Phenylarsine oxide and vanadate, inhibitors of protein-tyrosine phosphatases prevented the inhibitory effects of the cGMP and cAMP elevating agents on Ca(2+) entry and actin polymerization. These results suggest that Ca(2+) entry in human platelets is directly down-regulated by cGMP and cAMP by a mechanism involving the inhibition of cytoskeletal reorganization via the activation of protein tyrosine phosphatases.     

SNARE protein degradation upon platelet activation: calpain cleaves SNAP-23

In order to better understand the molecular mechanisms of platelet granule secretion, we evaluated the effect of activation-induced degranulation on three functional platelet SNARE proteins, SNAP-23, VAMP-3, and syntaxin 4. Initial studies showed that SNAP-23 is lost upon SFLLRN-induced platelet activation. Experiments with permeabilized platelets demonstrated that proteolysis of SNAP-23 was Ca(2+)-dependent. Ca(2+)-dependent proteolysis of SNAP-23 was inhibited by the cell-permeable calpain inhibitors, calpeptin and E-64d, as well as by the naturally occurring calpain inhibitor, calpastatin. In addition, purified calpain cleaved SNAP-23 in permeabilized platelets in a dose-dependent manner. In intact platelets, calpeptin prevented SFLLRN-induced degradation of SNAP-23. In contrast, calpeptin did not prevent SFLLRN-induced degradation of VAMP-3 and syntaxin 4 did not undergo substantial proteolysis following platelet activation. Calpain-induced cleavage of SNAP-23 was a late event occurring between 2.5 and 5 min following exposure of permeabilized platelets to Ca(2+). Experiments evaluating platelet alpha-granule secretion demonstrated that incubation of permeabilized platelets with 10 microM Ca(2+) prior to exposure to ATP inhibited ATP-dependent alpha-granule secretion from permeabilized platelets. SNAP-23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca(2+)-mediated degradation of SNAP-23 and reversed Ca(2+)-mediated inhibition of ATP-dependent alpha-granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP-23 and inhibits alpha-granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation.     

alpha(2A)-adrenergic receptor stimulation potentiates calcium release in platelets by modulating cAMP levels

alpha(2A)-Adrenergic receptor-mediated Ca(2+) signaling and integrin alpha(IIb)beta(3) exposure were investigated in human platelets under conditions where indirect, thromboxane- or ADP-mediated effects were absent. The alpha(2)-adrenergic receptor agonists, UK14304 and epinephrine (EPI), were unable to raise cytosolic levels of inositol 1,4,5-trisphosphate (InsP(3)) or Ca(2+) but potentiated the [Ca(2+)](i) rises evoked by other agonists that act through stimulation of phospholipase C (thrombin or platelet-activating factor) or stimulation of Ca(2+)-induced Ca(2+) release (CICR) in the absence of InsP(3) generation (thimerosal or thapsigargin). In addition, alpha(2)-adrenergic stimulation resulted in a 20% lowering in the cytosolic cAMP level. In platelets treated with G(salpha)-stimulating prostaglandin E(1), EPI increased the Ca(2+) signal evoked by either phospholipase C- or CICR-stimulating agonists mainly through modulation of the cAMP level. The stimulating effects of UK14304 and EPI on platelet Ca(2+) responses, and also on integrin alpha(IIb)beta(3) exposure and platelet aggregation, were abolished by pharmacological stimulation of cAMP-dependent protein kinase, and these effects were mimicked by inhibition of this activity. In permeabilized platelets, UK14304 and EPI potentiated InsP(3)-induced, CICR-mediated mobilization of Ca(2+) from internal stores in a similar way as did inhibition of cAMP-dependent protein kinase. In summary, a G(ialpha)-mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of alpha(2A)-adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the G(ialpha)-coupled alpha(2A)-adrenergic receptors does not result in phospholipase C activation but rather in modulation of the Ca(2+) response by relieving cAMP-mediated suppression of InsP(3)-dependent CICR.     

Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems

L-Arginine uptake and Ca(2+) changes in unstirred platelets activated by thrombin, collagen and Ca(2+) ionophore A23187 were evaluated. Thrombin did not affect L-arginine uptake at short incubation times (2-15 min), but at prolonged times slowed down the amino acid transport. Collagen was ineffective. A23187 decreased the L-arginine uptake in a dose-dependent manner, producing the maximal inhibition at 5 microM. In FURA 2-loaded platelets collagen did not modify Ca(2+) basal level, thrombin induced a late Ca(2+) rise and A23187 dose-dependently increased cytosolic Ca(2+), eliciting the highest increase at 5 microM. It is likely that L-arginine uptake is inversely modulated by Ca(2+) concentrations and is inhibited during platelet stimulation with agonists which induce cytosolic Ca(2+) elevation.     

Nitroprusside differentially inhibits ADP-stimulated calcium influx and mobilization in human platelets.

1. The effect of nitroprusside on cGMP concn., cAMP concn., shape change, aggregation, intracellular free Ca2+ concn. (by quin-2 fluorescence) and Mn2+ entry (by quenching of quin-2) was investigated in human platelets incubated with 1 mM-Ca2+ or 1 mM-EGTA. 2. Nitroprusside (10 nM-10 microM) caused similar concentration-dependent increases in platelet cGMP concn. and was without effect on cAMP concn. in the presence of extracellular Ca2+ or EGTA. 3. In ADP (3-6 microM)-stimulated platelets, nitroprusside caused 50% inhibition of shape change at 0.4 microM (+Ca2+) or 1.3 microM (+EGTA), aggregation at 0.09 microM (+Ca2+) and of increased intracellular Ca2+ at 0.02 microM (+Ca2+) or 2.1 microM (+EGTA). Entry of 1 mM-Mn2+ (-Ca2+) was inhibited by 80% by 5 microM-nitroprusside. 4. In ionomycin (20-500 nM)-stimulated platelets, nitroprusside (10 nM-100 microM) did not inhibit shape change or intracellular-Ca2+-increase responses, and only partially inhibited aggregation. 5. In phorbol myristate acetate (10 nM)-stimulated platelets, neither shape change nor aggregation was inhibited by 5 microM-nitroprusside. 6. The data demonstrate that nitroprusside inhibits ADP-mediated Ca2+ influx more potently than Ca2+ mobilization. Nitroprusside appears not to influence Ca2+ efflux or sequestration and not to affect the sensitivity of the activation mechanism to intracellular Ca2+ concn. or activation of protein kinase C.     

Identification of protein kinase Calpha as an essential, but not sufficient, cytosolic factor for Ca2+-induced alpha- and dense-core granule secretion in platelets

Upon activation, platelets release many active substances. Here, we have analyzed the mechanism governing Ca(2+)-induced secretion of von Willebrand factor stored in alpha-granules and 5-hydroxytryptamine in dense-core granules in permeabilized human platelets. Both secretions were dependent on ATP and cytosol. An essential factor for both granule secretions was purified from rat brain cytosol and identified to be protein kinase Calpha (PKCalpha) by partial amino acid sequencing. Purified PKCalpha efficiently stimulated both secretions in the presence of cytosol, whereas PKCalpha alone did not support the secretion of either type of granules, suggesting that PKCalpha is not a sufficient factor. Finally, in human platelet cytosol fractionated by a gel filtration column, the stimulatory activity for dense-core granule secretion paralleled with the concentration of PKC, suggesting that PKC could also be such a stimulatory factor in platelet cytosol. Thus, we identified PKCalpha as an essential, but not sufficient, cytosolic factor for the Ca(2+)-induced secretions of both alpha- and dense-core granules in platelets.     

Comparison of the role of protein kinase C in platelet functional responses induced by three different mechanisms, PAF, ionomycin and arachidonic acid.

The role of protein kinase C (PKC) in modulating platelet activation has been examined in platelets pre-incubated with either the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the non-specific protein kinase inhibitor, staurosporine. In order to determine where in the signal transduction pathway PKC is exerting its effect platelets were activated either with a receptor-operated stimulus platelet activating factor (PAF) or by direct elevation of [Ca2+]i (ionomycin) or with arachidonic acid which is converted into thromboxane B2 (TxB2). In PAF-stimulated platelets activation of PKC inhibited both [Ca2+]i elevation and TxB2 generation but had no effect on 5-hydroxytryptamine (5-HT) release whilst staurosporine increased the duration of [Ca2+]i elevation and potentiated TxB2 generation but inhibited 5-HT release. In ionomycin-stimulated platelets modulation of PKC had no effect on [Ca2+]i elevation but in contrast to PAF-stimulated platelets PKC activation caused potentiation of TxB2 generation and 5-HT release whilst inhibition of PKC caused inhibition of TxB2 generation and 5-HT release. Modulation of PKC did not affect arachidonic acid-induced TxB2 generation. These findings suggest that in receptor activated platelets endogenously activated PKC is exerting a negative feedback role, however, when [Ca2+]i elevation is not modified by PKC activation or inhibition (such as in ionomycin stimulated platelets) the relationship between the state of PKC activation and subsequent platelet functional responses corresponds more closely. The findings from this study suggest a different relationship between PKC and TxB2 generation than between PKC and dense granule release in PAF-stimulated platelets.     

Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets

Thrombin activates platelets in an ordered sequence of events that includes shape change, increase in cytoplasmic Ca(2+), activation of the alphaIIbbeta3 integrin, granule secretion, aggregation, and formation of a stable hemostatic plug. Activation of this process has also been implicated in the pathogenesis of atherosclerosis, stroke, and thrombosis. There are two identified thrombin-activated receptors on the surface of human platelets. PAR1 is a high-affinity thrombin receptor, and PAR4 is a low apparent affinity thrombin receptor of uncertain function. The goal of these studies is to determine the kinetics of thrombin activation of PAR1 and PAR4 and to relate the individual inputs from each receptor to platelet Ca(2+) signaling, secondary autocrine stimulation, and aggregation. Using a combination of PAR-specific peptide ligands and anti-PAR1 reagents, we separated the biphasic thrombin Ca(2+) response of platelets into two discrete components-a rapid spike response caused by PAR1, followed by a slower prolonged response from PAR4. Despite having a 20-70-fold slower rate of activation, PAR4 produces the majority of the integrated Ca(2+) signal that is sustained by the continuous presence of catalytically active thrombin. Surprisingly, PAR4 activation is much more effective than PAR1 activation in mounting secondary autocrine Ca(2+) signals from secreted ADP. The strong ADP response due to activated PAR4, however, requires prior activation of PAR1 as would normally occur during treatment of platelets with thrombin. Thus, the late signal generated by activated PAR4 is not redundant with the early signal from PAR1 and instead serves to greatly extend the high intracellular Ca(2+) levels that support the late phase of the platelet aggregation process.     

Bm-TFF2, a toad trefoil factor, promotes cell migration, survival and wound healing

Toad skin is naked and continually confronted by various injurious factors. Constant skin renewal and repairs occur frequently. However, the mechanisms of the renewal and repair have not clearly elucidated. In our previous work, a trefoil factor (TFF), Bm-TFF2, has been purified from the Bombina maxima skin and characterized as a platelet agonist. The mRNA of TFFs in toad skin was up-regulated greatly during the metamorphosis, indicating a pivotal role of TFFs in amphibian skin. Here, we presented the effects of Bm-TFF2 on the cell migration, apoptosis and proliferation. Bm-TFF2 bound to epithelial cells and showed strong cell motility activity. At the concentrations of 1-100 nM, Bm-TFF2-induced migration of human epithelial AGS and HT-29 cells, and rat intestinal epithelial IEC-6 cell lines. The in vitro wound healing assay also verified the activity of Bm-TFF2. Bm-TFF2 could also inhibit cell apoptosis induced by ceramide and sodium butyrate. The cell migration-promoting activity was abolished by MEK1 inhibitors, U0126 and PD98059, suggesting that ERK1/2 activation is crucial for Bm-TFF2 to stimulate cell migration. Taken together, Bm-TFF2 promoted wound healing by stimulating cell migration via MAPK pathway and preventing cell apoptosis. The potent biological activity of Bm-TFF2 makes it a useful molecular tool for further studies of structure-function relationship of the related human TFFs.     

Negative regulation of Gq-mediated pathways in platelets by G(12/13) pathways through Fyn kinase

Platelets contain high levels of Src family kinases (SFKs), but their functional role downstream of G protein pathways has not been completely understood. We found that platelet shape change induced by selective G(12/13) stimulation was potentiated by SFK inhibitors, which was abolished by intracellular calcium chelation. Platelet aggregation, secretion, and intracellular Ca(2+) mobilization mediated by low concentrations of SFLLRN or YFLLRNP were potentiated by SFK inhibitors. However, 2-methylthio-ADP-induced intracellular Ca(2+) mobilization and platelet aggregation were not affected by PP2, suggesting the contribution of SFKs downstream of G(12/13), but not G(q)/G(i), as a negative regulator to platelet activation. Moreover, PP2 potentiated YFLLRNP- and AYPGKF-induced PKC activation, indicating that SFKs downstream of G(12/13) regulate platelet responses through the negative regulation of PKC activation as well as calcium response. SFK inhibitors failed to potentiate platelet responses in the presence of G(q)-selective inhibitor YM254890 or in G(q)-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of G(q) pathways. Importantly, AYPGKF-induced platelet aggregation and PKC activation were potentiated in Fyn-deficient but not in Lyn-deficient mice compared with wild-type littermates. We conclude that SFKs, especially Fyn, activated downstream of G(12/13) negatively regulate platelet responses by inhibiting intracellular calcium mobilization and PKC activation through G(q) pathways.     

Dual role of platelet protein kinase C in thrombus formation: stimulation of pro-aggregatory and suppression of procoagulant activity in platelets

Protein kinase C (PKC) isoforms regulate many platelet responses in a still incompletely understood manner. Here we investigated the roles of PKC in the platelet reactions implicated in thrombus formation as follows: secretion aggregate formation and coagulation-stimulating activity, using inhibitors with proven activity in plasma. In human and mouse platelets, PKC regulated aggregation by mediating secretion and contributing to alphaIIbbeta3 activation. Strikingly, PKC suppressed Ca(2+) signal generation and Ca(2+)-dependent exposure of procoagulant phosphatidylserine. Furthermore, under coagulant conditions, PKC suppressed the thrombin-generating capacity of platelets. In flowing human and mouse blood, PKC contributed to platelet adhesion and controlled secretion-dependent thrombus formation, whereas it down-regulated Ca(2+) signaling and procoagulant activity. In murine platelets lacking G(q)alpha, where secretion reactions were reduced in comparison with wild type mice, PKC still positively regulated platelet aggregation and down-regulated procoagulant activity. We conclude that platelet PKC isoforms have a dual controlling role in thrombus formation as follows: (i) by mediating secretion and integrin activation required for platelet aggregation under flow, and (ii) by suppressing Ca(2+)-dependent phosphatidylserine exposure, and consequently thrombin generation and coagulation. This platelet signaling protein is the first one identified to balance the pro-aggregatory and procoagulant functions of thrombi.     

Protein kinase C alpha enhances sodium-calcium exchange during store-operated calcium entry in mouse platelets

A rise in intracellular calcium concentration ([Ca(2+)](i)) is necessary for platelet activation. A major component of the [Ca(2+)](i) elevation occurs through store-operated Ca(2+) entry (SOCE). The aim of this study was to understand the contribution of the classical PKC isoform, PKCα to platelet SOCE, using platelets from PKCα-deficient mice. SOCE was reduced by approximately 50% in PKCα(-/-) platelets, or following treatment with bisindolylmaleimide I, a PKC inhibitor. However, TG-induced Mn(2+) entry was unaffected, which suggests that divalent cation entry through store-operated channels is not directly regulated. Blocking the autocrine action of secreted ADP or 5-HT on its receptors did not reproduce the effect of PKCα deficiency. In contrast, SN-6, a Na(+)/Ca(2+) exchanger inhibitor, did reduce SOCE to the same extent as loss of PKCα, as did replacing extracellular Na(+) with NMDG(+). These treatments had no further effect in PKCα(-/-) platelets. These data suggest that PKCα enhances the extent of SOCE in mouse platelets by regulating Ca(2+) entry through the Na(+)/Ca(2+) exchanger.     

Collagen-induced platelet shape change is not affected by positive feedback pathway inhibitors and cAMP-elevating agents

Shape change is the earliest response of platelets to stimuli; it is mainly dependent upon Ca(2+)/calmodulin interaction subsequent to Ca(2+) mobilization and is mediated by myosin light chain kinase (MLCK) activation. It has been recently suggested that collagen itself is not able to elicit platelet shape change in the absence of ADP and thromboxane A(2) costimulation but is capable of inducing MLCK activation. Since we hypothesize that the morphological changes of the few platelets that adhere to collagen might not be revealed by turbidimetry, the aim of this study was to assess platelet shape change using transmission electron microscopy, in the absence of the amplificatory feedback pathways of ADP and thromboxane A(2). Our results demonstrated that only the platelets in contact with insoluble collagen fibers underwent a typical shape change, whereas those further away remained quiescent. Moreover, since cAMP enhances Ca(2+) mobilization in response to collagen, in the present study, we also investigated whether cAMP is involved in the inhibition of collagen-induced platelet shape change and MLC phosphorylation. Platelets were thus treated with iloprost (28 nm) prior to stimulation. Electron microscopy studies demonstrated that iloprost did not modify collagen-induced shape change, whereas immunoblotting studies showed a slight inhibition of MLC phosphorylation in the presence of enhanced cAMP levels. We can thus conclude that collagen is able to cause platelet shape change through activation of Ca(2+)/calmodulin-dependent MLCK, without the involvement of amplificatory pathways. Enhanced cytosolic cAMP levels do not inhibit collagen-induced platelet shape change but exert a weak inhibitory action on MLCK.     

Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C.

Low concentrations of Ca2+-mobilizing agonists such as vasopressin, platelet-activating factor, ADP, the endoperoxide analogue U44069 and the Ca2+ ionophore A23187 enhance the binding of [3H]phorbol 12,13-dibutyrate (PdBu) to intact human platelets. This effect is prevented by preincubation of platelets with prostacyclin (except for A23187). Adrenaline, which does not increase Ca2+ in the platelet cytosol, does not enhance the binding of [3H]PdBu to platelets. In addition, all platelet agonists except adrenaline potentiate the phosphorylation of the substrate of protein kinase C (40 kDa protein) induced by PdBu. Potentiation of protein kinase C activation is associated with increased platelet aggregation and secretion. Stimulus-induced myosin light-chain phosphorylation and shape change are not significantly affected, but formation of phosphatidic acid is decreased in the presence of PdBu. The results may indicate that low concentrations of agonists induce in intact platelets the translocation of protein kinase C to the plasma membrane by eliciting mobilization of Ca2+, and thereby place the enzyme in a strategic position for activation by phorbol ester. Such activation enhances platelet aggregation and secretion, but at the same time suppresses activation of phospholipase C. Therefore, at least part of the synergism evoked by Ca2+ and phorbol ester is mediated through a single pathway which involves protein kinase C. It is likely that the priming of protein kinase C by prior Ca2+ mobilization occurs physiologically in activated platelets.     

Phosphatidylinositol turnover in platelet activation; calcium mobilization and protein phosphorylation

Ca2+-activated, phospholipid-dependent protein kinase (C-kinase) in platelets is normally activated by diacylglycerol, which is derived from phosphatidylinositol through its receptor-linked breakdown. Under appropriate conditions this enzyme can also be activated by synthetic diacylglycerol which is directly added to intact platelets. C-Kinase thus activated preferentially phosphorylates an endogenous platelet protein having a molecular weight of approximately 40,000. This protein phosphorylation is merely a prerequisite but not a sufficient requirement for the release of serotonin. Evidence is presented suggesting that Ca2+ mobilization and C-kinase activation are synergistically involved in the physiological response of platelets to extracellular messengers, such as thrombin, collagen and platelet-activating factor.