Protein Kinase C�� Negatively Regulates Store-independent Ca2+ Entry and Phosphatidylserine Exposure Downstream of Glycoprotein VI in Platelets

Platelet activation must be tightly controlled to provide an effective, but not excessive, response to vascular injury. Cytosolic calcium is a critical regulator of platelet function, including granule secretion, integrin activation, and phosphatidylserine (PS) exposure. Here we report that the novel protein kinase C isoform, PKCθ, plays an important role in negatively regulating Ca²⁺ signaling downstream of the major collagen receptor, glycoprotein VI (GPVI). This limits PS exposure and so may prevent excessive platelet procoagulant activity. Stimulation of GPVI resulted in significantly higher and more sustained Ca²⁺ signals in PKCθ^−/− platelets. PKCθ acts at multiple distinct sites. PKCθ limits secretion, reducing autocrine ADP signaling that enhances Ca²⁺ release from intracellular Ca²⁺ stores. PKCθ thereby indirectly regulates activation of store-operated Ca²⁺ entry. However, PKCθ also directly and negatively regulates store-independent Ca²⁺ entry. This pathway, activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, was enhanced in PKCθ^−/− platelets, independently of ADP secretion. Moreover, LOE-908, which blocks 1-oleoyl-2-acetyl-sn-glycerol-induced Ca²⁺ entry but not store-operated Ca²⁺ entry, blocked the enhanced GPVI-dependent Ca²⁺ signaling and PS exposure seen in PKCθ^−/− platelets. We propose that PKCθ normally acts to restrict store-independent Ca²⁺ entry during GPVI signaling, which results in reduced PS exposure, limiting platelet procoagulant activity during thrombus formation.     

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.     

Non-redundant Roles of Phosphoinositide 3-Kinase Isoforms �� and �� in Glycoprotein VI-induced Platelet Signaling and Thrombus Formation

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cγ2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kα or -β strongly and selectively suppressed GPVI-induced Ca²⁺ mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cγ2 by PI3Kα/β. That PI3Kα and -β have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca²⁺ increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kα/β was blocked or p85α was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kδ or -γ. Furthermore, PI3Kα/β, but not PI3Kγ, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kα and -β isoforms are required for full GPVI-dependent platelet Ca²⁺ signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kα an interesting new target for anti-platelet therapy.     

Protein kinaseCdelta-calmodulin crosstalk regulates epidermal growth factor receptor exit from early endosomes

We have recently shown that calmodulin antagonist W13 interferes with the trafficking of the epidermal growth factor receptor (EGFR) and regulates the mitogen-activated protein kinase (MAPK) signaling pathway. In the present study, we demonstrate that in cells in which calmodulin is inhibited, protein kinase C (PKC) inhibitors rapidly restore EGFR and transferrin trafficking through the recycling compartment, although onward transport to the degradative pathway remains arrested. Analysis of PKC isoforms reveals that inhibition of PKCδ with rottlerin or its down-modulation by using small interfering RNA is specifically responsible for the release of the W13 blockage of EGFR trafficking from early endosomes. The use of the inhibitor Gö 6976, specific for conventional PKCs (α, β, and γ), or expression of dominant-negative forms of PKCλ, ζ, or ε did not restore the effects of W13. Furthermore, in cells treated with W13 and rottlerin, we observed a recovery of brefeldin A tubulation, as well as transport of dextran-fluorescein isothiocyanate toward the late endocytic compartment. These results demonstrate a specific interplay between calmodulin and PKCδ in the regulation of the morphology of and trafficking from the early endocytic compartment.     

Genetic Analysis of the Role of Protein Kinase Cθ in Platelet Function and Thrombus Formation

Background

PKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ^−/− T cells exhibit reduced activation and PKCθ^−/− mice are resistant to autoimmune disease, making PKCθ an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCθ positively regulates outside-in signalling through integrin α_IIbβ₃ in platelets, the role of PKCθ in GPVI-dependent signalling and functional activation of platelets has not been assessed.

Methodology/Principal Findings

In the present study we assessed static adhesion, cell spreading, granule secretion, integrin α_IIbβ₃ activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ^−/− platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ^−/− platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of α_IIbβ₃ activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s⁻¹) was enhanced.

Conclusions/Significance

These data suggest that PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and α_IIbβ₃ activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors.     

Differential Roles of the PKC Novel Isoforms,PKCδ and PKCε, in Mouse and Human Platelets

Background

Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation.

Methodology/Principal Findings

In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor.

Conclusions

These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI.     

Mechanism of Activation and Functional Role of Protein Kinase C�� in Human Platelets

The novel class of protein kinase C (nPKC) isoform η 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 nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y₁ receptor antagonist, or YM-254890, a G_q blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y₁ and G_q knock-out mice. However, pretreatment of platelets with P2Y₁₂ receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin α_IIbβ₃ mediated outside-in signaling. Moreover, in the presence of SC-57101, a α_IIbβ₃ receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, α_IIbβ₃ failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y₁ receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by α_IIbβ₃ integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis (1). Vascular injury exposes subendothelial collagen that activates platelets to change shape, secrete contents of granules, generate thromboxane, and finally aggregate via activated α_IIbβ₃ integrin, to prevent further bleeding (2, 3). ADP is a physiological agonist of platelets secreted from dense granules and is involved in feedback activation of platelets and hemostatic plug stabilization (4). It activates two distinct G-protein-coupled receptors (GPCRs) on platelets, P2Y₁ and P2Y₁₂, which couple to G_q and G_i, respectively (5–8). G_q activates phospholipase Cβ (PLCβ), which leads to diacyl glycerol (DAG)² generation and calcium mobilization (9, 10). On the other hand, G_i is involved in inhibition of cAMP levels and PI 3-kinase activation (4, 6). Synergistic activation of G_q and G_i proteins leads to the activation of the fibrinogen receptor integrin α_IIbβ₃. Fibrinogen bound to activated integrin α_IIbβ₃ further initiates feed back signaling (outside-in signaling) in platelets that contributes to the formation of a stable platelet plug (11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate various platelet functional responses such as dense granule secretion and integrin α_IIbβ₃ activation (12, 13). Based on their structure and cofactor requirements, PKCs are divided in to three classes: classical (cofactors: DAG, Ca²⁺), novel (cofactors: DAG) and atypical (cofactors: PIP₃) PKC isoforms (14). All the members of the novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ, η, and ε, are expressed in platelets (15), and they require DAG for activation. Among all the nPKCs, PKCδ (15, 16) and PKCθ (17–19) are fairly studied in platelets. Whereas nPKCδ is reported to regulate protease-activated receptor (PAR)-mediated dense granule secretion (15, 20), nPKCθ is activated by outside-in signaling and contributes to platelet spreading on fibrinogen (18). On the other hand, the mechanism of activation and functional role of nPKCη is not addressed as yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via three mechanisms (14, 21): 1) cofactor binding: upon cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as DAG, Ca²⁺, or PIP3, release autoinhibition, and attain an active conformation exposing catalytic domain of the enzyme. 2) phosphorylations: 3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates conserved threonine residues on activation loop of catalytic domain; this is followed by autophosphorylations of serine/threonine residues on turn motif and hydrophobic region. These series of phosphorylations maintain an active conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind receptors for activated C kinases (RACKs) and are lead to various subcellular locations to access the substrates (22, 23). Although various leading laboratories have elucidated the activation of PKCs, the mechanism of down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein phosphorylations by kinases and dephosphorylations by phosphatases has gained immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the serine/threonine phosphatases reported to date. Among them PP1 and PP2 phosphatases are known to regulate various platelet functional responses (24, 25). Furthermore, PP1c, is the catalytic unit of PP1 known to constitutively associate with α_IIb and is activated upon integrin engagement with fibrinogen and subsequent outside-in signaling (26). Among various PP1 isoforms, recently PP1γ is shown to positively regulate platelet functional responses (27). Thus, in this study we investigated if the above-mentioned phosphatases are involved in down-regulation of nPKCη. Furthermore, reports from other cell systems suggest that nPKCη regulates ERK/JNK pathways (28). In platelets ERK is known to regulate agonist induced thromboxane generation (29, 30). Thus, we also investigated if nPKCη regulates ERK phosphorylation and thereby agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP receptors and its inactivation by an integrin-associated phosphatase PP1γ. We also studied if nPKCη regulates functional responses in platelets and found that this isoform regulates ADP-induced thromboxane generation, but not fibrinogen receptor activation in platelets.     

Protein kinase C inhibitors alter neurotensin receptor binding and function in prostate cancer PC3 cells

Prostate cancer PC3 cells expressed constitutive protein kinase C (PKC) activity that under basal conditions suppressed neurotensin (NT) receptor function. The endogenous PKC activity, assessed using a cell-based PKC substrate phosphorylation assay, was diminished by PKC inhibitors and enhanced by phorbol myristic acid (PMA). Accordingly, PKC inhibitors (staurosporine, Go-6976, Go-6983, Ro-318220, BIS-1, chelerythrine, rottlerin, quercetin) enhanced NT receptor binding and NT-induced inositol phosphate (IP) formation. In contrast, PMA inhibited these functions. The cells expressed conventional PKCs (, βI) and novel PKCs (δ, ε), and the effects of PKC inhibitors on NT binding were blocked by PKC downregulation. The inhibition of NT binding by PMA was enhanced by okadaic acid and blocked by PKC inhibitors. However, when some PKC inhibitors (rottlerin, BIS-1, Ro-318220, Go-69830, quercetin) were used at higher concentrations (> 2 μM), they had a different effect characterized by a dramatic increase in NT binding and an inhibition of NT-induced IP formation. The specificity of the agents implicated novel PKCs in this response and indeed, the inhibition of NT-induced IP formation was reproduced by PKCδ or PKCε knockdown. The inhibition of IP formation appeared to be specific to NT since it was not observed in response to bombesin. Scatchard analyses indicated that the PKC-directed agents modulated NT receptor affinity, not receptor number or receptor internalization. These findings suggest that PKC participates in heterologous regulation of NT receptor function by two mechanisms: a) — conventional PKCs inhibit NT receptor binding and signaling; and b) — novel PKCs maintain the ability of NT to stimulate PLC. Since NT can activate PKC upon binding to its receptor, it is possible that NT receptor is also subject to homologous regulation by PKC.     

Characterization of the Differential Roles of the Twin C1a and C1b Domains of Protein Kinase C��

Classic and novel protein kinase C (PKC) isozymes contain two zinc finger motifs, designated “C1a” and “C1b” domains, which constitute the recognition modules for the second messenger diacylglycerol (DAG) or the phorbol esters. However, the individual contributions of these tandem C1 domains to PKC function and, reciprocally, the influence of protein context on their function remain uncertain. In the present study, we prepared PKCδ constructs in which the individual C1a and C1b domains were deleted, swapped, or substituted for one another to explore these issues. As isolated fragments, both the δC1a and δC1b domains potently bound phorbol esters, but the binding of [³H]phorbol 12,13-dibutyrate ([³H]PDBu) by the δC1a domain depended much more on the presence of phosphatidylserine than did that of the δC1b domain. In intact PKCδ, the δC1b domain played the dominant role in [³H]PDBu binding, membrane translocation, and down-regulation. A contribution from the δC1a domain was nonetheless evident, as shown by retention of [³H]PDBu binding at reduced affinity, by increased [³H]PDBu affinity upon expression of a second δC1a domain substituting for the δC1b domain, and by loss of persistent plasma membrane translocation for PKCδ expressing only the δC1b domain, but its contribution was less than predicted from the activity of the isolated domain. Switching the position of the δC1b domain to the normal position of the δC1a domain (or vice versa) had no apparent effect on the response to phorbol esters, suggesting that the specific position of the C1 domain within PKCδ was not the primary determinant of its activity.One of the essential steps for protein kinase C (PKC)² activation is its translocation from the cytosol to the membranes. For conventional (α, βI, βII, and γ) and novel (δ, ε, η, and θ) PKCs, this translocation is driven by interaction with the lipophilic second messenger sn-1,2-diacylglycerol (DAG), generated from phosphatidylinositol 4,5-bisphosphate upon the activation of receptor-coupled phospholipase C or indirectly from phosphatidylcholine via phospholipase D (1). A pair of zinc finger structures in the regulatory domain of the PKCs, the “C1” domains, are responsible for the recognition of the DAG signal. The DAG-C1 domain-membrane interaction is coupled to a conformational change in PKC, both causing the release of the pseudosubstrate domain from the catalytic site to activate the enzyme and triggering the translocation to the membrane (2). By regulating access to substrates, PKC translocation complements the intrinsic enzymatic specificity of PKC to determine its substrate profile.The C1 domain is a highly conserved cysteine-rich motif (∼50 amino acids), which was first identified in PKC as the interaction site for DAG or phorbol esters (3). It possesses a globular structure with a hydrophilic binding cleft at one end surrounded by hydrophobic residues. Binding of DAG or phorbol esters to the C1 domain caps the hydrophilic cleft and forms a continuous hydrophobic surface favoring the interaction or penetration of the C1 domain into the membrane (4). In addition to the novel and classic PKCs, six other families of proteins have also been identified, some of whose members possess DAG/phorbol ester-responsive C1 domains. These are the protein kinase D (5), the chimaerin (6), the munc-13 (7), the RasGRP (guanyl nucleotide exchange factors for Ras and Rap1) (8), the DAG kinase (9), and the recently characterized MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) families (10). Of these C1 domain-containing proteins, the PKCs have been studied most extensively and are important therapeutic targets (11). Among the drug candidates in clinical trials that target PKC, a number such as bryostatin 1 and PEP005 are directed at the C1 domains of PKC rather than at its catalytic site.Both the classic and novel PKCs contain in their N-terminal regulatory region tandem C1 domains, C1a and C1b, which bind DAG/phorbol ester (12). Multiple studies have sought to define the respective roles of these two C1 domains in PKC regulation, but the issue remains unclear. Initial in vitro binding measurements with conventional PKCs suggested that 1 mol of phorbol ester bound per mole of PKC (13-15). On the other hand, Stubbs et al., using a fluorescent phorbol ester analog, reported that PKCα bound two ligands per PKC (16). Further, site-directed mutagenesis of the C1a and C1b domains of intact PKCα indicated that the C1a and C1b domains played equivalent roles for membrane translocation in response to phorbol 12-myristate 13-acetate (PMA) and (-)octylindolactam V (17). Likewise, deletion studies indicated that the C1a and C1b domains of PKCγ bound PDBu equally with high potency (3, 18). Using a functional assay with PKCα expression in yeast, Shieh et al. (19) deleted individual C1 domains and reported that C1a and C1b were both functional and equivalent upon stimulation by PMA, with either deletion causing a similar reduction in potency of response, whereas for mezerein the response depended essentially on the C1a domain, with much weaker response if only the C1b domain was present. Using isolated C1 domains, Irie et al. (20) suggested that the C1a domain of PKCα but not those of PKCβ or PKCγ bound [³H]PDBu preferentially; different ligands showed a generally similar pattern but with different extents of selectivity. Using synthesized dimeric bisphorbols, Newton''s group reported (21) that, although both C1 domains of PKCβII are oriented for potential membrane interaction, only one C1 domain bound ligand in a physiological context.In the case of novel PKCs, many studies have been performed on PKCδ to study the equivalency of the twin C1 domains. The P11G point mutation of the C1a domain, which caused a 300-fold loss of binding potency in the isolated domain (22), had little effect on the phorbol ester-dependent translocation of PKCδ in NIH3T3 cells, whereas the same mutation of the C1b caused a 20-fold shift in phorbol ester potency for inducing translocation, suggesting a major role of the C1b domain for phorbol ester binding (23). A secondary role for the C1a domain was suggested, however, because mutation in the C1a domain as well as the C1b domain caused a further 7-fold shift in potency. Using the same mutations in the C1a and C1b domains, Bögi et al. (24) found that the binding selectivity for the C1a and C1b domains of PKCδ appeared to be ligand-dependent. Whereas PMA and the indole alkaloids indolactam and octylindolactam were selectively dependent on the C1b domain, selectivity was not observed for mezerein, the 12-deoxyphorbol 13-monoesters prostratin and 12-deoxyphorbol 13-phenylacetate, and the macrocyclic lactone bryostatin 1 (24). In in vitro studies using isolated C1a and C1b domains of PKCδ, Cho''s group (25) described that the two C1 domains had opposite affinities for DAG and phorbol ester; i.e. the C1a domain showed high affinity for DAG and the C1b domain showed high affinity for phorbol ester. No such difference in selectivity was observed by Irie et al. (20).PKC has emerged as a promising therapeutic target both for cancer and for other conditions, such as diabetic retinopathy or macular degeneration (26-30). Kinase inhibitors represent one promising approach for targeting PKC, and enzastaurin, an inhibitor with moderate selectivity for PKCβ relative to other PKC isoforms (but still with activity on some other non-PKC kinases) is currently in multiple clinical trials. An alternative strategy for drug development has been to target the regulatory C1 domains of PKC. Strong proof of principle for this approach is provided by multiple natural products, e.g. bryostatin 1 and PEP005, which are likewise in clinical trials and which are directed at the C1 domains. A potential advantage of this approach is the lesser number of homologous targets, <30 DAG-sensitive C1 domains compared with over 500 kinases, as well as further opportunities for specificity provided by the diversity of lipid environments, which form a half-site for ligand binding to the C1 domain. Because different PKC isoforms may induce antagonistic activities, inhibition of one isoform may be functionally equivalent to activation of an antagonistic isoform (31).Along with the benzolactams (20, 32), the DAG lactones have provided a powerful synthetic platform for manipulating ligand: C1 domain interactions (31). For example, the DAG lactone derivative 130C037 displayed marked selectivity among the recombinant C1a and C1b domains of PKCα and PKCδ as well as substantial selectivity for RasGRP relative to PKCα (33). Likewise, we have shown that a modified DAG lactone (dioxolanones) can afford an additional point of contact in ligand binding to the C1b domain of PKCδ (34). Such studies provide clear examples that ligand-C1 domain interactions can be manipulated to yield novel patterns of recognition. Further selectivity might be gained with bivalent compounds, exploiting the spacing and individual characteristics of the C1a and C1b domains (35). A better understanding of the differential roles of the two C1 domains in PKC regulation is critical for the rational development of such compounds. In this study, by molecularly manipulating the C1a or C1b domains in intact PKCδ, we find that both the C1a and C1b domains play important roles in PKCδ regulation. The C1b domain is predominant for ligand binding and for membrane translocation of the whole PKCδ molecule. The C1a domain of intact PKCδ plays only a secondary role in ligand binding but stabilizes the PKCδ molecule at the plasma membrane for downstream signaling. In addition, we show that the effect of the individual C1 domains of PKCδ does not critically depend on their position within the regulatory domain.     

Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow

Background

The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function.

Objectives

Our aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo.

Methods and Results

We studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa.

Conclusions

Endocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function may prove beneficial in the search for new antithrombotic therapies.     

Vitamin E isoforms differentially regulate intercellular adhesion molecule-1 activation of PKCα in human microvascular endothelial cells

Aims

ICAM-1-dependent leukocyte recruitment in vivo is inhibited by the vitamin E isoform d-α-tocopherol and elevated by d-γ-tocopherol. ICAM-1 is reported to activate endothelial cell signals including protein kinase C (PKC), but the PKC isoform and the mechanism for ICAM-1 activation of PKC are not known. It is also not known whether ICAM-1 signaling in endothelial cells is regulated by tocopherol isoforms. We hypothesized that d-α-tocopherol and d-γ-tocopherol differentially regulate ICAM-1 activation of endothelial cell PKC.

Results

ICAM-1 crosslinking activated the PKC isoform PKCα but not PKCβ in TNFα-pretreated human microvascular endothelial cells. ICAM-1 activation of PKCα was blocked by the PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, ERK1/2 inhibitor PD98059, and xanthine oxidase inhibitor allopurinol. ERK1/2 activation was blocked by inhibition of XO and PLC but not by inhibition of PKCα, indicating that ERK1/2 is downstream of XO and upstream of PKCα during ICAM-1 signaling. During ICAM-1 activation of PKCα, the XO-generated ROS did not oxidize PKCα. Interestingly, d-α-tocopherol inhibited ICAM-1 activation of PKCα but not the upstream signal ERK1/2. The d-α-tocopherol inhibition of PKCα was ablated by the addition of d-γ-tocopherol.

Conclusions

Crosslinking ICAM-1 stimulated XO/ROS which activated ERK1/2 that then activated PKCα. ICAM-1 activation of PKCα was inhibited by d-α-tocopherol and this inhibition was ablated by the addition of d-γ-tocopherol. These tocopherols regulated ICAM-1 activation of PKCα without altering the upstream signal ERK1/2. Thus, we identified a mechanism for ICAM-1 activation of PKC and determined that d-α-tocopherol and d-γ-tocopherol have opposing regulatory functions for ICAM-1-activated PKCα in endothelial cells.     

Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation

Background

We have previously shown the presence of a TRAF4/p47^phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.

Aims

To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.

Methods and Results

Human and mouse washed platelets (from WT or Pyk2 KO mice) were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively) and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P) surface expression) and integrin α_IIbβ₃ activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk), PI3-K and Bruton''s tyrosine kinase (Btk) and upstream of Rac1, PLCγ2, Ca²⁺ release, PKC, Hic-5, NOX1 and α_IIbβ₃ activation.

Conclusion

Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.     

UTP-dependent inhibition of Na+ absorption requires activation of PKC in endometrial epithelial cells

The objective of this study was to investigate the mechanism of uridine 5′-triphosphate (UTP)-dependent inhibition of Na⁺ absorption in porcine endometrial epithelial cells. Acute stimulation with UTP (5 μM) produced inhibition of sodium absorption and stimulation of chloride secretion. Experiments using basolateral membrane–permeabilized cell monolayers demonstrated a reduction in benzamil-sensitive Na⁺ conductance in the apical membrane after UTP stimulation. The UTP-dependent inhibition of sodium transport could be mimicked by PMA (1 μM). Several PKC inhibitors, including GF109203X and Gö6983 (both nonselective PKC inhibitors) and rottlerin (a PKCδ selective inhibitor), were shown to prevent the UTP-dependent decrease in benzamil-sensitive current. The PKCα-selective inhibitors, Gö6976 and PKC inhibitor 20–28, produced a partial inhibition of the UTP effect on benzamil-sensitive Isc. Inhibition of the benzamil-sensitive Isc by UTP was observed in the presence of BAPTA-AM (50 μM), confirming that activation of PKCs, and not increases in [Ca²⁺]_i, were directly responsible for the inhibition of apical Na⁺ channels and transepithelial Na⁺ absorption.     

Characterisation of the Ral GTPase inhibitor RBC8 in human and mouse platelets

The Ral GTPases, RalA and RalB, have been implicated in numerous cellular processes, but are most widely known for having regulatory roles in exocytosis. Recently, we demonstrated that deletion of both Ral genes in a platelet-specific mouse gene knockout caused a substantial defect in surface exposure of P-selectin, with only a relatively weak defect in platelet dense granule secretion that did not alter platelet functional responses such as aggregation or thrombus formation. We sought to investigate the function of Rals in human platelets using the recently described Ral inhibitor, RBC8. Initial studies in human platelets confirmed that RBC8 could effectively inhibit Ral GTPase activation, with an IC₅₀ of 2.2 μM and 2.3 μM for RalA and RalB, respectively. Functional studies using RBC8 revealed significant, dose-dependent inhibition of platelet aggregation, secretion (α- and dense granule), integrin activation and thrombus formation, while α-granule release of platelet factor 4, Ca²⁺ signalling or phosphatidylserine exposure were unaltered. Subsequent studies in RalAB-null mouse platelets pretreated with RBC8 showed dose-dependent decreases in integrin activation and dense granule secretion, with significant inhibition of platelet aggregation and P-selectin exposure at 10 μM RBC8. This study strongly suggests therefore that although RBC8 is useful as a Ral inhibitor in platelets, it is likely also to have off-target effects in the same concentration range as for Ral inhibition. So, whilst clearly useful as a Ral inhibitor, interpretation of data needs to take this into account when assessing roles for Rals using RBC8.     

Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.     

Reduction of β-Amyloid Levels by Novel Protein Kinase C? Activators

Isoform-specific protein kinase C (PKC) activators may be useful as therapeutic agents for the treatment of Alzheimer disease. Three new ϵ-specific PKC activators, made by cyclopropanation of polyunsaturated fatty acids, have been developed. These activators, AA-CP4, EPA-CP5, and DHA-CP6, activate PKCϵ in a dose-dependent manner. Unlike PKC activators that bind to the 1,2-diacylglycerol-binding site, such as bryostatin and phorbol esters, which produce prolonged down-regulation, the new activators produced sustained activation of PKC. When applied to cells expressing human APPSwe/PS1δ, which produce large quantities of β-amyloid peptide (Aβ), DCP-LA and DHA-CP6 reduced the intracellular and secreted levels of Aβ by 60–70%. In contrast to the marked activation of α-secretase produced by PKC activators in fibroblasts, the PKC activators produced only a moderate and transient activation of α-secretase in neuronal cells. However, they activated endothelin-converting enzyme to 180% of control levels, suggesting that the Aβ-lowering ability of these PKCϵ activators is caused by increasing the rate of Aβ degradation by endothelin-converting enzyme and not by activating nonamyloidogenic amyloid precursor protein metabolism.     

Novel Antileukemic Compound Ingenol 3-Angelate Inhibits T Cell Apoptosis by Activating Protein Kinase C��

Members of the protein kinase C (PKC) family of serine-threonine kinases are important regulators of immune cell survival. Ingenol 3-angelate (PEP005) activates a broad range of PKC isoforms and induces apoptosis in acute myeloid leukemia cells by activating the PKC isoform PKCδ. We show here that, in contrast to its effect on leukemic cells, PEP005 provides a strong survival signal to resting and activated human T cells. The antiapoptotic effect depends upon the activation of PKCθ. This PKC isoform is expressed in T cells but is absent in myeloid cells. Further studies of the mechanism involved in this process showed that PEP005 inhibited activated CD8⁺ T cell apoptosis through the activation of NFκB downstream of PKCθ, leading to increased expression of the antiapoptotic proteins Mcl-1 and Bcl-x_L. Transfection of CD8⁺ T cells with dominant-negative PKCθ diminished the prosurvival effect of PEP005 significantly. Ectopic expression of PKCθ in the acute myeloid leukemia cell line NB4 turned their response to PEP005 from an increased to decreased rate of apoptosis. Therefore, in contrast to myeloid leukemia cells, PEP005 provides a strong survival signal to T cells, and the expression of functional PKCθ influences whether PKC activation leads to an anti- or proapoptotic outcome in the cell types tested.     

Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (~15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.