Calcium tolerant rabbit cardiomyocytes, isolated by collagenase perfusion, were preincubated for varying periods of time followed by resuspension in fresh media and centrifugation into an ischaemic pellet with restricted extracellular fluid. Pellets were incubated for 240 min under oil at 37°C to mimic severe ischaemia. Time to onset of ischaemic contracture (rod to square transformation) and trypan blue permeability following resuspension in 85 mOsm media were monitored at sequential times. The protocol of Series 1 was a 5-10 min pre-incubation, immediately followed by ischaemic pelleting. Preincubation with pinacidil (50 μm) protected cells from ischaemic insult, but pinacidil added only into the ischaemic pellet did not protect. Protection was abolished by the protein kinase (PKC) inhibitors chelerythrine (10 μm) added with pinacidil and calphostin C (200nm) added only into the ischaemic pellet. Neither PKC inhibitor had an effect on injury of untreated ischaemic myocytes (data not shown). Series 2-5 were preconditioning protocols with a 10 min intervention period, followed by a 30 min oxygenated drug-free period, prior to ischaemic pelleting. In series 2 pinacidil protected cells from ischaemic insult and this protection was abolished when glyburide (10 μm) was present during preincubation, or during post-incubation and ischaemia. Glyburide only partially inhibited the protection when glyburide was added only into the ischaemic pellet. In Series 3, 8-sulfophenyltheophyline (SPT)(100 μm) or adenosine deaminase during preincubation, or SPT only added into the ischaemic pellet abolished pinacidil’s protection. In Series 4, cardiomyocytes were ischaemically preconditioned by pelleting for 10 min followed by 30 min reoxygenation. Glyburide during initial ischaemic blocked protection, but when added during post incubation and into the final pellet protection was not reduced. In Series 5 8-cyclopentyl-1,3, dipropylxanthine (DPCPX) (10 μm) added into the final pellet abolished protection by pinacidil, but not protection following ischaemic preconditioning. In contrast to pinacidil, ischaemically preconditioned cells maintain protection in the presence of glyburide, indicating that: (1) pinacidil does not exactly mimic preconditioning and (2) ischaemically preconditioned cells do not require opened K+ATP channels for protection, although they appear to be important during initiation of the preconditioned state. It is hypothesized that pinacidil opening of K+ channels may facilitate induction of preconditioning.

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Platelets are primary components of hemostasis. However, incongruous activation of platelets lead to thrombosis, which result in multiple cardio-vascular and cerebrovascular complications. Thus, platelet activation is tightly regulated. Molecular components that aid in activation of platelets have been extensively studied. However, molecular pathways that negatively regulate platelet activation and prevent accidental activation of platelets are poorly understood. In this study we investigated the molecular mechanisms that negatively regulate platelet activation. Protein Kinase C isforms (PKCs) are serine threonine kinases that regulate various platelet functional responses leading to hemostasis. Positive regulatory role of PKCs towards platelet aggregation and secretion has been extensively studied. However, we have recently demonstrated that PKCs negatively regulate ADP- induced thromboxane generation. The PKC isoforms and mechanism involved in this process have not been known. Thus, in this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms that regulate thromboxane generation. Thromboxane generation in platelets is a multi-step process beginning with cPLA2 activation. cPLA2 activation is the rate limiting step in the process of thromboxane generation. Furthermore, cPLA2 activation is regulated by ERK and calcium in various cell systems including platelets. PKC inhibition potentiated both cPLA2 and ERK activation, suggesting that PKCs negatively regulate thromboxane generation by regulating ERK activation, which in turn regulates cPLA2 activation. Furthermore, we have also shown that PKCs negatively regulate ADP-induced calcium mobilization. ADP activates platelets via P2Y1 and P2Y12 receptors. P2Y12 receptor-mediated signaling is shown to positively regulate P2Y1-mediated calcium mobilization in platelets. Furthermore, PKCs are shown to negatively regulate P2Y12 receptor desensitization in platelets. Thus, we investigated if PKCs regulate calcium mobilization indirectly by regulating P2Y12 receptor function. However, PKCs regulate calcium mobilization independent of P2Y12 receptor signaling. In summary we have shown that PKC isoforms negatively regulate ADP-induced thromboxane generation by regulating calcium mobilization and ERK activation that in turn regulates cPLA2 activity. We further investigated the PKC isoforms involved in this process. Based on our results with Go-6976, a classical PKC inhibitor and GF109203X, a pan PKC inhibitor, we identified that that novel or atypical PKC isoforms are involved in negative regulation of ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We first investigated the nPKCs activated by ADP. In aspirin-treated platelets, ADP failed to activate nPKC θ and δ non-stirring conditions. Thus, we conclude that these isoforms are not involved in negative regulation of thromboxane generation. We further investigated if other non-classical PKC isoforms i. e nPKC η and ε or atypical PKC isoforms could be involved in this process. We began our investigation with the mechanism of activation and functional role of nPKC η in platelets. The mechanism of activation of PKCs has been extensively studied in various cell systems including platelets. However, the mechanism by which they are inactivated is not completely understood. In this study, we demonstrate a novel mechanism of inactivation of nPKC η isoform by integrin associated serine/threonine phosphatase. we demonstrated that ADP activates nPKC η via P2Y1 receptor coupled to Gq. As expected, Gi pathway, which does not generate DAG or mobilize calcium, has no role in regulation of nPKC η. Interestingly, we show that upon activation of platelets, αIIbβ3 mediated outside-in signaling dephosphorylates nPKCη through PP1γ phosphatase. We have also evaluated the role of nPKC η using η-RACK antagonistic peptides that interfere with enzyme-substrate interaction. Similar antagonistic peptides have been successfully used in various cell systems such as cardiomyocytes and neuronal cell. Using η-RACK antagonists we have demonstrated that nPKC η positively regulates agonist- induced thromboxane generation with no effect on agonist- induced platelet aggregation. The peptides were targeted in to the cell using TAT carrier protein, which is also used as a negative control for these experiments. The specificity of η-RACK antagonistic peptides is further elucidated by the fact that they do not affect the platelet aggregation. In summary, nPKC η is activated by ADP via P2Y1 receptor and is dephosphorylated by integrin αIIbβ3 via PP1γ phosphatase. Furthermore, activated nPKC η positively regulates ADP- induced thromboxane generation with no effect on aggregation. Since, our aim was to investigate the nPKC isoforms that negatively regulate ADPinduced thromboxane generation we investigated if nPKC ε is involved in this process. We made use of PKC ε knockout mice (PKC ε KO) for this process. We observed potentiated thromboxane generation in ADP-induced PKC ε murine platelets compared to witd type murine platelets. Thus, PKC ε might be one of the PKC isoforms involved in negative regulation of ADP-induced thromboxane generation. However, we failed to detect PKC ε in human platelets using western blot analysis. Since, PKC ε has been reported to be a part of platelet kinase repertoire, it could be limitation of our technique that we failed to detect it in western blot analysis. Since, PKCs negatively regulate ADP-induced thromboxane generation, we also investigated if PKCs also regulate PAR-mediated thromboxane generation. Similar to ADP, PAR-mediated thromboxane generation is not affected by Classical PKC isoforms. Furthermore, although non-classical PKC isoforms negatively regulate thromboxane generation, the extent of negative regulation is smaller and non-significant compared to ADP. Thus, we investigated if activation of nPKC isoforms were different between ADP and AYPGKF (PAR4 agonist). While, ADP fails to activate nPKC δ and θ, PARs activate Them. Furthermore, we have recently demonstrated that nPKC δ and θ are positive regulators of PAR-mediated platelet functional responses. Therefore, PKCinduced potentiation of thromboxane generation by ADP and PAR agonist are different due to differential activation of PKCs. This data lead to our final project, where we investigated the reason for differential activation of nPKC isoforms by various platelet agonists. Using strong and weak platelet agonists and DAG analogue, DiC8, we demonstrated that different platelet agonists differentially regulate nPKC activation due to variable amounts of DAG generated by them. Furthermore, we also have demonstrated that nPKC η and ε have higher affinities to DAG compared to nPKC δ and θ. / Molecular and Cellular Physiology

Protein Kinase C delta (PKCδ) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. We evaluated the role of PKCδ in platelets using two approaches - pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (δV1-1)TAT, and in the murine platelets lacking PKCδ, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKCδ. However, PKCδ had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKCδ null mouse platelets and in human platelets pretreated with (δV1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKCδ-/- mice occluded similar to their wild-type littermates. Hence, we conclude that PKCδ differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKCδ deficiency does not affect the thrombus formation in vivo. We further investigated the mechanism of such differential regulation of dense granule release by PKCδ in platelets. SH2 domain-containing Inositol Phosphatase (SHIP)-1 is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists, SFLLRN and AYPGKF, or GPVI agonist, convulxin. GPVImediated SHIP-1 phosphorylation occurred rapidly at 15 sec whereas PAR-mediated phosphorylation was delayed, occurring at 1 min. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKCδ upon stimulation of platelets with a GPVI agonists, but not with a PAR agonist. In PKCδ null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited, suggesting that PKCδ regulates the phosphorylation of SHIP-1. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1, Y311 and Y155 of PKCδ were inhibited. In murine platelets lacking Lyn, or SHIP-1, GPVI-mediated dense granule secretions were potentiated, whereas PAR-mediated dense granule secretions were inhibited. Phosphorylated SHIP-1 associated with phosphorylated-Y155 PKCδ peptide. Therefore, we conclude that Lyn-mediated phosphorylations of PKCδ and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets. / Physiology

Platelets are crucial components of the hemostatic machinery of the body. When the endothelial continuity is disrupted due to injury or atherosclerotic plaque rupture, one of the earliest responses to arrest the bleeding is the adhesion of circulating platelets to the exposed subendothelial collagen matrix. Subsequent intracellular signaling mediated downstream of various receptor systems leads to alpha IIb beta 3 activation, thromboxane generation, ADP release, etc., culminating in platelet clot or thrombus formation. The protein kinase family of enzymes mediates a significant number of these intracellular signaling events that culminate in platelet activation. These enzymes can be broadly classified into two classes- tyrosine kinases and serine/threonine kinases. Syk (spleen tyrosine kinase) is an important non-receptor tyrosine kinase present in platelets and plays an important role downstream of GPVI-FcR gamma chain receptor complex activation. We studied the effects of curcumin (diferuloylmethane), which is the active ingredient found in the herbal remedy and food spice turmeric, on the GPVI-mediated platelet activation. We have found that it significantly inhibits the kinase activity of Syk without affecting its phosphorylation. Pre-incubating the platelets with curcumin for only a minute resulted in a concentration-dependent inhibition of aggregation and secretion, with approximately 75% inhibition observed at 50 mM curcumin. Additionally, the activation-dependent phosphorylation of tyrosines 753/759 on PLC gamma2 and phosphorylation of tyrosine 191 on the transmembrane scaffold protein LAT, were inhibited (p<0.05). However, the phosphorylation of the activation loop tyrosines 525/526 on Syk and of the tyrosine 145 on intracellular adaptor molecule SLP-76 were not significantly affected. Furthermore, the inhibitory action of curcumin on the catalytic activity of Syk was independent of any of its effects on the thromboxane generation because all our studies were performed using aspirin-treated platelets. PKC zeta is an atypical member of the PKC family of serine/threonine kinases. In this study, we have confirmed that it is expressed in human platelets and is constitutively phosphorylated at the activation loop threonine 410 as well as the turn motif threonine 560, which is an autophosphorylation site. Phosphorylation at these two residues has been shown to be important for its kinase activity. Furthermore, agonist-mediated platelet aggregation under stirring condition results in dephosphorylation of the Thr410 residue, which can be prevented by blocking integrin alpha IIb beta 3 by its antagonist SC-57101 (p<0.01). The dephosphorylation of Thr410 can also be prevented by okadaic acid, a Ser/Thr protein phosphatase inhibitor, at concentrations above 100 nM. However, in PP1c gamma null mice, we did not observe any effect on the dephosphorylation, suggesting that other isoforms of PP1 or other classes of the phosphatases could be responsible for this phenomenon, at least in these knockout mice. The basal phosphorylation of Thr560, however, remained unaffected by agonist stimulation, integrin activation, integrin blockade, okadaic acid treatment and in the PP1c gamma null mice. It can be speculated that PKC zeta may be constitutively active under basal resting conditions and acts as a negative regulator of platelet activation or functional responses. The Thr560 autophosphorylation signal alone may not be sufficient to sustain its full enzymatic activity. / Physiology

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