Soochong Kim

Platelet purinergic receptors.

Activation of P2Y(1) and P2Y(12) receptors, through secreted ADP that is stimulated by agonists such as thrombin, thromboxane and collagen, is a major mechanism of platelet activation. P2X(1) receptors also participate in platelet shape change and potentiation of calcium mobilization. The cloning of the P2Y(12) receptor and its subsequent knockout in mice promises further understanding of its downstream signaling events.

Role of Phosphoinositide 3-Kinase β in Glycoprotein VI-mediated Akt Activation in Platelets

Glycoprotein (GP) VI is a critical platelet collagen receptor. Phosphoinositide 3-kinase (PI3K) plays an important role in GPVI-mediated platelet activation, yet the major PI3K isoforms involved in this process have not been identified. In addition, stimulation of GPVI results in the activation of Akt, a downstream effector of PI3K. Thus, we investigated the contribution of PI3K isoforms to GPVI-mediated platelet activation and Akt activation. A protein kinase C inhibitor GF 109203X or a P2Y12 receptor antagonist AR-C69931MX partly reduced GPVI-induced Akt phosphorylation. Platelets from mice dosed with clopidogrel also showed partial Akt phosphorylation, indicating that GPVI-mediated Akt phosphorylation is regulated by both secretion-dependent and -independent pathways. In addition, GPVI-induced Akt phosphorylation in the presence of ADP antagonists was completely inhibited by PI3K inhibitor LY294002 and PI3Kβ inhibitor TGX-221 indicating an essential role of PI3Kβ in Akt activation directly downstream of GPVI. Moreover, GPVI-mediated platelet aggregation, secretion, and intracellular Ca2+ mobilization were significantly inhibited by TGX-221, and less strongly inhibited by PI3Kα inhibitor PIK75, but were not affected by PI3Kγ inhibitor AS252424 and PI3Kδ inhibitor IC87114. Consistently, GPVI-induced integrin αIIbβ3 activation of PI3Kγ−/− and PI3Kδ−/− platelets also showed no significant difference compared with wild-type platelets. These results demonstrate that GPVI-induced Akt activation in platelets is dependent in part on Gi stimulation through P2Y12 receptor activation by secreted ADP. In addition, a significant portion of GPVI-dependent, ADP-independent Akt activation also exists, and PI3Kβ plays an essential role in GPVI-mediated platelet aggregation and Akt activation.

P2Y12 receptor‐mediated potentiation of thrombin‐induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation

Summary.  Background: Thromboxane A2 (TXA2) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA2. The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin‐induced TXA2 generation. Results: PAR1‐activating peptide (SFLLRN), PAR4‐activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (AA) from membrane‐bound phospholipids, and subsequent TXA2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y12 receptor antagonist, AR‐C69931MX, but not the P2Y1 receptor antagonist, MRS2179. In addition, AYPGKF‐ and thrombin‐induced TXA2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA2 when compared with the wild‐type littermates in response to PAR stimulation. Inhibition of extracellular signal‐regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR‐mediated cPLA2 phosphorylation and TXA2 generation. Further, platelets that were pretreated with AR‐C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists. Conclusions: Based on these findings, we conclude that thrombin‐induced Erk1/2 activation is essential for PAR‐mediated TXA2 generation, which is potentiated by the P2Y12 receptor‐mediated signaling pathway but not the P2Y1 receptor‐mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR‐mediated Src activation precedes Erk1/2 activation.

ADP receptors--targets for developing antithrombotic agents.

Platelet P2 receptors--P2Y1, P2Y12, and P2X1--constitute the means by which adenine nucleotides can activate platelets. Coactivation of the Galphaq-coupled P2Y1 and Galphai2-coupled P2Y12 receptors is necessary for ADP-mediated platelet activation, which forms the basis of using P2 antagonists as antithrombotic drugs. P2Y1 receptor antagonists inhibit platelet activation, while P2Y1 knockout mice show longer bleeding times than normal mice but few other problems; however, its ubiquitous expression in other tissues renders P2Y1 questionable as an antithrombotic target. The P2Y12 receptor is expressed nearly exclusively in platelets and brain, making it an attractive antithrombotic target. Antagonists for the P2Y12 receptor have been developed that either require metabolic activation to covalently inhibit P2Y12 and are irreversible, or simply are competitive in nature and thus reversible. Ticlopidine and clopidogrel are irreversible P2Y12 antagonists and have been repeatedly proven as clinical antithrombotic agents. In addition, a recently reported P2Y12 antagonist, CS-747, shows promise as a future antithrombotic drug. The AR-C series of compounds represent reversible P2Y12 antagonists and have been used extensively to characterize the function of P2Y12 in platelets. Clinical studies show that AR-C69931MX is as effective as clopidogrel; furthermore, the combination of AR-C69931MX (cangrelor) and clopidogrel confers greater antagonism of P2Y12 than either antagonist alone. The P2X1 receptor is a calcium channel that functions to potentiate agonist-induced platelet shape change, and its inhibition or loss has little if any effect on hemostasis. A combination of P2Y1 and P2Y12 antagonists may represent an additional course of antithrombotic treatment.

Impaired activation of platelets lacking protein kinase C-θ isoform

Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-theta is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC-theta-selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PAR-induced aggregation, dense and alpha-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-theta, platelet aggregation and secretion were also impaired. PKC-mediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-theta RACK peptide, which may contribute to the lower levels of granule secretion when PKC-theta function is lost. Furthermore, the level of JON/A binding to activated alpha(IIb)beta(3) receptor was also significantly decreased in PKC-theta(-/-) mice compared with wild-type littermates. PKC-theta(-/-) murine platelets showed significantly lower agonist-induced thromboxane A(2) (TXA(2)) release through reduced extracellular signal-regulated kinase phosphorylation. Finally, PKC-theta(-/-) mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl(3) in vivo thrombosis model compared with wild-type mice. In conclusion, PKC-theta isoform plays a significant role in platelet functional responses downstream of PAR and GPVI receptors.

Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets.

We have previously shown that ADP-induced thromboxane generation in platelets requires signalling events from the G(q)-coupled P2Y1 receptor (platelet ADP receptor coupled to stimulation of phospholipase C) and the G(i)-coupled P2Y12 receptor (platelet ADP receptor coupled to inhibition of adenylate cyclase) in addition to outside-in signalling. While it is also known that extracellular calcium negatively regulates ADP-induced thromboxane A2 generation, the underlying mechanism remains unclear. In the present study we sought to elucidate the signalling mechanisms and regulation by extracellular calcium of ADP-induced thromboxane A2 generation in platelets. ERK (extracllular-signal-regulated kinase) 2 activation occurred when outside-in signalling was blocked, indicating that it is a downstream event from the P2Y receptors. However, blockade of either P2Y1 or the P2Y12 receptors with corresponding antagonists completely abolished ERK phosphorylation, indicating that both P2Y receptors are required for ADP-induced ERK activation. Inhibitors of Src family kinases or the ERK upstream kinase MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] abrogated ADP-induced ERK phosphorylation and thromboxane A2 generation. Finally ADP- or G(i)+G(z)-induced ERK phosphorylation was blocked in the presence of extracellular calcium. The present studies show that ERK2 is activated downstream of P2Y receptors through a complex mechanism involving Src kinases and this plays an important role in ADP-induced thromboxane A2 generation. We also conclude that extracellular calcium blocks ADP-induced thromboxane A2 generation through the inhibition of ERK activation.

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms alpha, beta, gamma and delta in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kbeta-selective inhibitor, but not by PIK75 (a PI3Kalpha inhibitor), AS252424 (a PI3Kgamma inhibitor) or IC87114 (a PI3Kdelta inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1-/- mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kbeta in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kbeta plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kbeta mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

Lipid rafts are required in Gαi signaling downstream of the P2Y12 receptor during ADP‐mediated platelet activation

Summary.  ADP is important in propagating hemostasis upon its secretion from activated platelets in response to other agonists. Lipid rafts are microdomains within the plasma membrane that are rich in cholesterol and sphingolipids, and have been implicated in the stimulatory mechanisms of platelet agonists. We sought to determine the importance of lipid rafts in ADP‐mediated platelet activation via the G protein‐coupled P2Y1 and P2Y12 receptors using lipid raft disruption by cholesterol depletion with methyl‐β‐cyclodextrin. Stimulation of cholesterol‐depleted platelets with ADP resulted in a reduction in the extent of aggregation but no difference in the extent of shape change or intracellular calcium release. Furthermore, repletion of cholesterol to previously depleted membranes restored ADP‐mediated platelet aggregation. In addition, P2Y12‐mediated inhibition of cAMP formation was significantly decreased upon cholesterol depletion from platelets. Stimulation of cholesterol‐depleted platelets with agonists that depend upon Gαi activation for full activation displayed significant loss of aggregation and secretion, but showed restoration when simultaneously stimulated with the Gαz‐coupled agonist epinephrine. Finally, Gαi preferentially localizes to lipid rafts as determined by sucrose density centrifugation. We conclude that Gαi signaling downstream of P2Y12 activation, but not Gαq or Gαz signaling downstream of P2Y1 or α2A activation, respectively, has a requirement for lipid rafts that is necessary for its function in ADP‐mediated platelet activation.

Lyn, PKC-δ, SHIP-1 interactions regulate GPVI-mediated platelet-dense granule secretion

Protein kinase C-delta (PKC-delta) is expressed in platelets and activated downstream of protease-activated receptors (PARs) and glycoprotein VI (GPVI) receptors. We have previously shown that PKC-delta positively regulates PAR-mediated dense granule secretion, whereas it negatively regulates GPVI-mediated dense granule secretion. We further investigated the mechanism of such differential regulation of dense granule release by PKC-delta in platelets. SH2 domain-containing inositol phosphatase-1 (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. GPVI-mediated SHIP-1 phosphorylation occurred rapidly at 15 seconds, whereas PAR-mediated phosphorylation was delayed, occurring at 1 minute. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKC-delta on stimulation of platelets with a GPVI agonist, but not with a PAR agonist. In PKC-delta-null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1 and Y311 of PKC-delta were inhibited. In murine platelets lacking Lyn or SHIP-1, GPVI-mediated dense granule secretions are potentiated, whereas PAR-mediated dense granule secretions are inhibited. Therefore, we conclude that Lyn-mediated phosphorylations of PKC-delta and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets.

Different G protein‐coupled signaling pathways are involved in α granule release from human platelets

Summary.  Alpha granule release plays an important role in propagating a hemostatic response upon platelet activation. We evaluated the ability of various agonists to cause α granule release in platelets. Alpha granule release was measured by determining P‐selectin surface expression in aspirin‐treated washed platelets. ADP‐induced P‐selectin expression was inhibited both by MRS 2179 (a P2Y1 selective antagonist) and AR‐C69931MX (a P2Y12 selective antagonist), suggesting a role for both Gαq and Gαi pathways in ADP‐mediated α granule release. Consistent with these observations, the combination of serotonin (a Gαq pathway stimulator) and epinephrine (a Gαz pathway stimulator) also caused α granule release. Furthermore, U46619‐induced P‐selectin expression was unaffected by MRS 2179 but was dramatically inhibited by AR‐C69931, indicating a dominant role for P2Y12 in U46619‐mediated α granule release. Additionally, the Gα12/13‐stimulating peptide YFLLRNP potentiated α granule secretion in combination with either ADP or serotonin/epinephrine costimulation but was unable to induce secretion by itself. Finally, costimulation of the Gαi and Gα12/13 pathways resulted in a significant dose‐dependent increase in α granule release. We conclude that ADP‐induced α granule release in aspirin‐treated platelets occurs through costimulation of Gαq and Gαi signaling pathways. The P2Y12 receptor plays an important role in thromboxane A2‐mediated α granule release, and furthermore activation of Gα12/13 and Gαq signaling pathway can cause α granule release.