Activation of Protein Kinase C Is Required for the Stable Attachment of Adherent Platelets to Collagen but Is Not Needed for the Initial Rapid Adhesion Under Flow Conditions (original) (raw)
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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.
Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen
Journal of Biological …, 2010
Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.
Blood, 2005
Thrombin has been known to cause tyrosine phosphorylation of protein kinase C δ (PKCδ) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCδ and the role of this event in platelet function. PKCδ was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCδ did not occur in Gαq-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5′-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ′, N ′-tetraacetic acid), suggesting a role for Gαq pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyro...
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.
The EMBO Journal, 1996
Thrombin-stimulated aggregation of human platelets promotes an increase in the phosphatidylinositol 4-phosphate (Ptdlns 4-P) 5-kinase (PIPkin) activity in the cytoskeleton. This phenomenon is associated with translocation of PIPkin isoform C to the cytoskeleton and with an increase in the amount of phosphatidylinositol bisphosphate (PtdInsP2) bound to the cytoskeletal pellet. All three of these effects are prevented if the platelets are not stirred or if RGD-containing peptides are present, demonstrating that they require integrin activation. All three are also abolished by pretreatment with okadaic acid, which also prevents the aggregation-dependent translocation of pp6OC-src to the cytoskeleton. The results point to the existence of a cytoskeletally associated PtdInsP2 pool under the control of integrin-mediated signals that act via PIPkin C and suggest that a common, okadaic acid-sensitive mechanism may underlie the aggregation-dependent translocation of certain signalling molecules to the platelet cytoskeleton.
The Biochemical journal, 1988
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 tr...
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.