Swaminathan Murugappan

Nucleotide receptor signaling in platelets

Summary.  Upon injury to a vessel wall the exposure of subendothelial collagen results in the activation of platelets. Platelet activation culminates in shape change, aggregation, release of granule contents and generation of lipid mediators. These secreted and generated mediators trigger a positive feedback mechanism potentiating the platelet activation induced by physiological agonists such as collagen and thrombin. Adenine nucleotides, adenosine diphosphate (ADP) and adenosine triphosphate (ATP), released from damaged cells and that are secreted from platelet‐dense granules, contribute to the positive feedback mechanism by acting through nucleotide receptors on the platelet surface. ADP acts through two G protein‐coupled receptors, the Gq‐coupled P2Y1 receptor, and the Gi‐coupled P2Y12 receptor. ATP, on the other hand, acts through the ligand‐gated channel P2X1. Stimulation of platelets by ADP leads to shape change, aggregation and thromboxane A2 generation. ADP‐induced dense granule release depends on generated thromboxane A2. Furthermore, costimulation of both P2Y1 and P2Y12 receptors is required for ADP‐induced platelet aggregation. ATP stimulation of P2X1 is involved in platelet shape change and helps to amplify platelet responses mediated by agonists such as collagen. Activation of each of these nucleotide receptors results in unique signal transduction pathways that are important in the regulation of thrombosis and hemostasis.

Differential Role of Protein Kinase Cδ Isoform in Agonist-induced Dense Granule Secretion in Human Platelets

Several platelet agonists, including thrombin, collagen, and thromboxane A2, cause dense granule release independently of thromboxane generation. Because protein kinase C (PKC) isoforms are implicated in platelet secretion, we investigated the role of individual PKC isoforms in platelet dense granule release. PKCδ was phosphorylated in a time-dependent manner that coincided with dense granule release in response to protease-activated receptor-activating peptides SFLLRN and AYPGKF in human platelets. Only agonists that caused platelet dense granule secretion activated PKCδ. SFLLRN- or AYPGKF-induced dense granule release and PKCδ phosphorylation occurred at the same respective agonist concentration. Furthermore, AYPGKF and SFLLRN-induced dense granule release was blocked by rottlerin, a PKCδ selective inhibitor. In contrast, convulxin-induced dense granule secretion was potentiated by rottlerin but was abolished by Go6976, a classical PKC isoform inhibitor. However, SFLLRN-induced dense granule release was unaffected in the presence of Go6976. Finally, rottlerin did not affect SFLLRN-induced platelet aggregation, even in the presence of dimethyl-BAPTA, indicating that PKCδ has no role in platelet fibrinogen receptor activation. We conclude that PKCδ and the classical PKC isoforms play a differential role in platelet dense granule release mediated by protease-activated receptors and glycoprotein VI. Furthermore, PKCδ plays a positive role in protease-activated receptor-mediated dense granule secretion, whereas it functions as a negative regulator downstream of glycoprotein VI signaling.

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.

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.

Protein Kinase Cδ Differentially Regulates Platelet Functional Responses

Objective—Protein Kinase C delta (PKC&dgr;) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. The purpose of this study was to investigate the role of PKC&dgr; in platelets. Methods and Results—We evaluated the role of PKC&dgr; in platelets using two approaches—pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (&dgr; V1-1)TAT, and in the murine platelets lacking PKC&dgr;, 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 PKC&dgr;. However, PKC&dgr; 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 PKC&dgr; null mouse platelets and in human platelets pretreated with (&dgr; V1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKC&dgr;−/− mice occluded similar to their wild-type littermates. Conclusions—Hence, we conclude that PKC&dgr; differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKC&dgr; deficiency does not affect the thrombus formation in vivo.

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.

Differential regulation of threonine and tyrosine phosphorylations on protein kinase Cdelta by G-protein-mediated pathways in platelets.

Phosphorylation of activation loop threonine (Thr(505)) and regulatory domain tyrosine (Tyr(311)) residues are key regulators of PKC (protein kinase C) delta function in platelets. In the present study, we show that G(q) and G(12/13) pathways regulate the Thr(505) and Tyr(311) phosphorylation on PKCdelta in an interdependent manner. DiC8 (1,2-dioctanoylglycerol), a synthetic analogue of DAG (diacylglycerol), caused Thr(505), but not Tyr(311), phosphorylation on PKCdelta, whereas selective activation of G(12/13) pathways by the YFLLRNP peptide failed to cause phosphorylation of either residue. However, simultaneous activation by DiC8 and YFLLRNP resulted in Thr(505) and Tyr(311) phosphorylation on PKCdelta. In addition, we found that the activation of SFKs (Src family tyrosine kinases) is essential for G(12/13)-mediated Tyr(311) phosphorylation of PKCdelta. These results were confirmed using G(q)-deficient mouse platelets. Finally, we investigated whether Thr(505) phosphorylation is required for Tyr(311) phosphorylation. A T505A PKCdelta mutant failed to be phosphorylated at Tyr(311), even upon stimulation of both G(q) and G(12/13) pathways. We conclude that (i) PKCdelta binding to DAG, downstream of G(q) pathways, and its translocation results in Thr(505) phosphorylation, (ii) G(12/13) pathways activate SFKs required for the phosphorylation of Tyr(311) on Thr(505)-phosphorylated PKCdelta, and (iii) Thr(505) phosphorylation is a prerequisite for Tyr(311) phosphorylation on PKCdelta.

Clopidogrel: interactions with the P2Y12 receptor and clinical relevance.

Adenosine diphosphate (ADP) activates platelets through binding to two G protein coupled receptors on the platelet, the P2Y1 receptor that couples to Gq, and the P2Y12 receptor that couples to Gi pathways. Stimulation of both receptors is necessary to cause ADP-induced GPIIb/IIIa activation [1–3]; however the P2Y12 receptor has also been implicated in the potentiation of responses caused by other agonists such as thromboxane A2, thrombin, and collagen. Clopidogrel (commercial name: Plavix) is a clinically effective irreversible antagonist for the P2Y12 receptor, and many studies have focused on characterizing the effect of P2Y12 antagonism on platelet aggregation and in the prevention of thrombosis in patients at risk for ischemic events. The cloning of the P2Y12 receptor has occurred relatively recently [4,6] compared with the time that P2Y12 receptor antagonists have been effective in the clinic. While the mechanism of interaction between the P2Y12 receptor and metabolites of clopidogrel has previously been speculative, recent studies have focused on the interaction between residues on the P2Y12 receptor and these anti-platelet agents. The use of thiol reagents that selectively antagonize the P2Y12 receptor has led to the identification of specific residues on the P2Y12 receptor that are of importance to this interaction. P2Y12 antagonism has proven benefits in the clinic. Knowledge of the specific interaction between the P2Y12 receptor and clopidogrel or related compounds would then serve as a basis for future improvements on compounds that target the P2Y12 receptor for prevention of thrombosis. This review will outline the structure of the P2Y12 receptor, discuss the interactions between clopidogrel and the P2Y12 receptor, and provide an overview of the benefits of P2Y12 antagonism as a therapeutic target.