Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1991
We studied the action of the az adrenergic agonist adrenaline on the platelet responses evoked by the activation of protein kinase C or by the ionophore induced increase of cytosolic Ca 2+. Both the phorbol ester and ionomycin-induced aggregation are strongly potentiated by adrenaline which per se does not behave as an activating agonist. The potentiation by adrenaline is observed both when added before and after the aggregating agent; in the latter case the effect increases on increasing the delay of ~'.dren~ine addition. Adrenaline also reverses the inhibition by cAMP of the PMA (or ionomycin) induced aggregation. It also has a strong potentiating effect (over 100%) on the phorbol ester induced ATP secretion and a weaker effect on the secretion induced by ionomycin. The effect on secretion is visible only when adrenaline is added prior to the stimulus. The inhibition by cAMP of the PMA or ionomycin induced secretion is also counteracted by adrenaline. In no case adrenaline modifies the pattern of platelet phosphoproteins. lonomycin induces some platelet aggregation also in the presence of the protein kinase inhibitor staurosporine; also this phosphoprotein independent aggregation is strongly stimulated by adrenaline.
Biochemical Journal, 1990
The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositolcontaining phospholipids relied upon the separation of the products [3H]inositol mono-, bisand tris-phosphates by Dowex-l chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone HI in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95 % of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 ,ug/ml) did not enhance phosphoinositide production, and at higher concentrations (50 ,ug/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation. Abbreviations used: cAMP, cyclic AMP (adenosine 3',5' monophosphate); CPT-cAMP, 8-chlorophenylthio-cAMP; DAG, diacylglycerol; Gprotein; GTP-binding protein; IP, inositol monophosphate; IP2, inositol bisphosphate; IP3, inositol trisphosphate; NP40, Nonidet P-40; p47, 40-47 kDa protein; PAF, platelet-activating factor; PGI2, prostacyclin (prostaglandin 12); PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5bisphosphate; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PKIP, cAMP-dependent protein kinase inhibitor peptide; PS, phosphatidylserine; TPA, 12-O-tetradecanoylphorbol 13-acetate.
Correlation between cytosolic Ca2+ concentration, protein phosphorylation and platelet secretion
Cell Calcium, 1996
Addition of the calcium-ionophore ionomycin to acetylsalicylate-treated platelets suspended in a low Ca*+ concentration-containing medium (about 0.1 PM), induced a dose-dependent (range 0.25-3 ,uM) and transient increase in the cytosolic Caz+ concentration ([Ca*+],). Less than 10% of the maximal releasable amount of serotonin was secreted at [Ca2+], lower than 1 PM, whereas secretion was almost maximal at [Ca*+], higher than 2 PM. In all cases the secretion stopped after about 1 min even if the [Ca'+], was kept constant by repeated small additions of CaCI, (25-40 yM). A rapid phosphorylation of pleckstrin (47 kDa) and myosin light chain (20 kDa) was found in all cases, whereas a weak phosphorylation of a 27 kDa protein occurred at [Ca2+], lower than 1.5 yM. Addition of 0.2 mM CaCI, to platelets pretreated for 4 min with 0.5-l uM ionomycin brought about a serotonin secretion remarkably lower than that obtained by the simultaneous addition of CaCI, and ionophore. Platelets suspended in a low calcium-containing medium and exposed to ionomycin showed a major increase in tyrosine phosphorylation of 60 and 72 kDa proteins and a slight increment in tyrosine phosphorylation of 115 and 130 kDa proteins. Subsequent addition of 0.2 mM CaCI, induced a widespread phosphotyrosine dephosphorylation, particularly evident in the 60 kDa protein identified as p60 CSrC kinase. The protein kinase inhibitor genistein caused, together with a marked prevention of the protein tyrosine phosphorylation, a remarkable increase in the ionomycinelicited secretory activity of platelets. All together these results indicate that protein kinase C-dependent pleckstrin phosphorylation is a prerequisite of platelet secretion, but that the latter process is apparently regulated by a network of phosphoproteins, in particular the serine/threonine phosphorylation of 27 and 68 kDa proteins and the tyrosine phosphorylation of the p60c-"'" were found to be associated with a decrease in the secretory activity.
Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets
Journal of Biological Chemistry, 2009
The novel class of protein kinase C (nPKC) isoform is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKC using pharmacological and gene knockout approaches. nPKC was phosphorylated (at Thr-512) in a time-and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y 1 receptor antagonist, or YM-254890, a G q blocker, abolished 2MeSADP-induced phosphorylation of nPKC. Similarly, ADP failed to activate nPKC in platelets isolated from P2Y 1 and G q knockout mice. However, pretreatment of platelets with P2Y 12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKC phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKC was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin ␣ IIb  3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a ␣ IIb  3 receptor antagonist, nPKC dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1c␥, a catalytic subunit of serine/threonine phosphatase, ␣ IIb  3 failed to dephosphorylate nPKC. Thus, we conclude that ADP activates nPKC via P2Y 1 receptor and is subsequently dephosphorylated by PP1␥ phosphatase activated by ␣ IIb  3 integrin. In addition, pretreatment of platelets with-RACK antagonistic peptides, a specific inhibitor of nPKC, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKC positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.
Blood, 2001
Protein kinase C (PKC)–potentiated inhibitory phosphoprotein of myosin phosphatase (CPI) was detected in human platelets. Like smooth muscle CPI-17, in vitro phosphorylation of platelet CPI by PKC inhibited the activity of myosin phosphatase containing the PP1δ catalytic subunit and the 130-kd myosin-binding subunit (MBS). Treatment of intact platelets with thrombin or the stable thromboxane A2 analog STA2 resulted in increased phosphorylation of both CPI and MBS at Thr-696, whereas phorbol myristate acetate (PMA) and the Ca++ ionophore ionomycin only induced CPI phosphorylation. PMA induced slow adenosine triphosphate (ATP) secretion of fura 2–loaded platelets with no change in cytosolic Ca++. The PMA-induced increase in CPI phosphorylation preceded phosphorylation of 20-kd myosin light chain (MLC20) at Ser-19 and ATP secretion. The PKC inhibitor, GF109203X, inhibited PMA-induced phosphorylation of CPI and MLC20 with similar IC50 values. These findings suggest that the activation o...
Biochemical Journal, 1992
Protein kinase C (PKC) acts in synergy with Ca2+ mobilization for the activation of platelets. Three different PKC subtypes that specifically react with antibodies to alpha- beta- and zeta-PKC have been detected in human platelets. We have compared the subcellular redistribution of these isoforms in platelets after exposure to the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and to two physiological agonists, thrombin and vasopressin. In the presence of PMA, beta-PKC is most rapidly translocated to membranes, followed by zeta-PKC and alpha-PKC [membrane contents of 39 +/- 6, 31 +/- 4 and 24 +/- 4% (means +/- S.E.M.) respectively after 2 min incubation]. In contrast, both thrombin and vasopressin induced a biphasic translocation of PKC isoforms. For both agonists, the first phase of translocation occurred within 1 min and was identical for the three isoforms. However, during the second phase, the translocation of zeta-PKC by thrombin and vasopressin differed [...
Phorbol esters sensitize platelets to activation by physiological agonists
Blood, 1987
Phorbol esters such as phorbol 12, 13-dibutyrate (PdBu; 40 to 200 nmol/L) or 12-O-tetradecanoyl phorbol 13-acetate (20 to 80 nmol/L) added to aspirinized platelet-rich plasma (PRP) 5 to 15 seconds prior to various platelet stimuli (epinephrine, ADP, prostaglandin endoperoxide analog U44069, collagen, PAF, or vasopressin) potentiate the rate and extent of aggregation and ATP secretion induced by those agonists. Platelet aggregation, but not secretion, is potentiated at low concentrations of agonists; platelet secretion is potentiated at higher concentrations of the platelet stimuli. Potentiation of platelet responses was also observed when the preincubation time with PdBu was extended to 12 minutes and also occurred in washed platelets. The potentiating effect of phorbol esters is not mediated by formation of arachidonate metabolites or by released ADP. The sensitizing effect of PdBu on platelet aggregation induced by epinephrine is unique, since in contrast to the other platelet sti...
Spacial isolation of protein kinase C activation in thrombin stimulated human platelets
Biochemical and Biophysical Research Communications, 1988
Thrombin stimulation of human platelets is associated with turnover of inositol phospholipids, mobilization of intracellular Ca 2+ 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 ~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 a2-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.
InflammoPharmacology, 2001
This study was conducted to examine the mechanism(s) of synergistic interaction of adrenaline and platelet-activating factor (PAF) mediated human platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5-8 nM) plus adrenaline (0.5-2 ¹M) was inhibited by both ® 2-adrenoceptor blocker (yohimbine) and PAF receptor antagonist (WEB2086). While examining the role of the downstream signalling pathways, we found that this synergism was inhibited by calcium channel blockers, verapamil, and diltiazem. In addition, platelet aggregation by co-addition of adrenaline and PAF was also inhibited by very low concentrations of phospholipase C (PLC) inhibitor (U73122; IC 50 D 0:2 ¹M), the MAP kinase inhibitor, PD98059 (IC 50 D 3 ¹M) and cyclooxygenase (COX-1) inhibitors including indomethacin (IC 50 D 0:25 ¹M), urbiprofen (IC 50 D 0:7 ¹M) and piroxicam (IC 50 D 7 ¹M). However, the COX-2 inhibitor, nimesulide, was also effective in inhibiting the aggregation. The inhibitors of tyrosine kinase (genistien) and phosphatidylinositol 3-kinase inhibitor (wortmannin) had no signi cant effects on platelet aggregation. These data suggest that the synergistic effect of adrenaline and PAF on human platelet aggregation is receptor-mediated and involves the activation of PLC /calcium, COX and MAP kinase signalling pathways.
Cell Calcium, 2015
Rises in cytosolic Ca 2+ concentration ([Ca 2+ ] cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca 2+ ] cyt , but these only monitor the net effect of manipulations on the processes controlling [Ca 2+ ] cyt (Ca 2+ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca 2+ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca 2+ signalling, we here monitor Ca 2+ flux around the platelet by measuring net Ca 2+ fluxes to or from the extracellular space and the intracellular Ca 2+ stores, which act as the major sources and sinks for Ca 2+ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na + concentration ([Na + ] cyt), which influences platelet Ca 2+ fluxes via Na + /Ca 2+ exchange. The intracellular store Ca 2+ concentration ([Ca 2+ ] st) was monitored using Fluo-5N, the extracellular Ca 2+ concentration ([Ca 2+ ] ext) was monitored using Fluo-4 whilst [Ca 2+ ] cyt and [Na + ] cyt were monitored using Fura-2 and SFBI respectively. PKC inhibition using Ro-31-8220 or bisindolaemide I potentiated ADP-and thrombin-evoked rises in [Ca 2+ ] cyt in the absence of extracellular Ca 2+. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca 2+ release and Ca 2+ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca 2+ ] cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na + ] cyt which would be expected to reduce Ca 2+ removal via the Na + /Ca 2+ exchanger (NCX). Thrombinevoked rises in [Na + ] cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn 2+ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca 2+ ] cyt following SERCA inhibition and either removal of extracellular Na + or inhibition of Na + /K +-ATPase activity by removal of extracellular K + or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca 2+ ] cyt by acceleration of SERCA activity, whilst rises in [Ca 2+ ] cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na + /K +-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na + ] cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca 2+ signalling.