Haripriya Shankar

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.

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.

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.

Ligand directed signaling differences between rodent and human κ-opioid receptors.

Background: Dysphoric effects of κ-opioid receptor (KOR) agonists require p38 MAPK activation in mice, but sequence differences in human KOR may affect this mechanism. Results: Differences in p38 activation were observed between human and rodent KOR for pentazocine and butorphanol. Conclusion: Species differences affect signaling. Significance: Rodent models may not predict adverse effects of KOR agonists in humans. KOR activation of Gβγ dependent signaling results in analgesia, whereas the dysphoric effects of KOR agonists are mediated by a different pathway involving G protein receptor kinase and non-visual arrestin. Based on this distinction, a partial KOR agonist that does not efficiently activate arrestin-dependent biased signaling may produce analgesia without dysphoria. No KOR-selective partial agonists are currently available, and preclinical assessment is complicated by sequence differences between rodent (r) and human (h) KOR. In this study, we compared the signaling initiated by the available partial agonists. Pentazocine was significantly more potent at activating p38 MAPK in hKOR than rKOR expressed in HEK293 cells but equally potent at arrestin-independent activation of ERK1/2 in hKOR and rKOR. Similarly, butorphanol increased phospho-p38-ir in hKOR-expressing cells but did not activate p38 in rKOR-HEK293. Like pentazocine, butorphanol was equally efficacious at activating ERK1/2 in rKOR and hKOR. In contrast, levorphanol, nalorphine, and U50,488 did not distinguish between hKOR and rKOR in p38 MAPK activation. Consistent with its low potency at p38 activation, pentazocine did not produce conditioned place aversion in mice. hKOR lacks the Ser-369 phosphorylation site in rKOR required for G protein receptor kinase/arrestin-dependent p38 activation, but mutation of the Ser-358 to asparagine in hKOR blocked p38 activation without affecting the acute arrestin-independent activation of ERK1/2. This study shows that hKOR activates p38 MAPK through a phosphorylation and arrestin-dependent mechanism; however, activation differs between hKOR and rKOR for some ligands. These functional selectivity differences have important implications for preclinical screening of partial KOR agonists.

G-protein dependent platelet signaling--perspectives for therapy.

Platelet activation and aggregation is an integral component of the pathophysiology that leads to thrombotic and ischemic diseases such as cerebral stroke, peripheral vascular disease and myocardial infarction. Anti-platelet agents (such as aspirin, ADP receptor antagonists, and GPIIb/IIIa antagonists), phosphodiesterase inhibitors and anti-coagulants are major part of the current treatment towards treating ischemic diseases. However, their limited efficacy in the setting of arterial thrombosis, unfavorable side effect profile and cost-to-benefit issues substantiate the need for the development of newer and more efficacious antithrombotic drugs. Various platelet agonists like adenosine diphosphate (ADP), thrombin and thromboxane A2 (TXA2) activate platelets by acting via their respective surface receptors, which couple to one or more distinct G-proteins belonging to either the G(i), G(q), G(12/13) or G(s) families. Upon activation, each of these G-proteins trigger a series of intracellular signaling cascades, causing the platelets to undergo shape change, secrete their granular contents, generate positive feedback mediators and form stable platelet aggregates. In addition, various G-protein-mediated signaling cascades act in synergy with one another to amplify the magnitude of the platelet responses. The significance of G-proteins as key mediators of the platelet function and normal hemostasis is further corroborated by extensive gene knockout studies. In this review we will limit our discussion to understanding the role of G-proteins in the process of platelet activation and discuss some of the anti-thrombotic drugs that mediate their beneficial effects by interfering with or preventing the initiation of the G-protein signaling pathway.

Hematopoeitic lineage cell-specific protein-1 (HS1) regulates PAR-mediated ERK activation and thromboxane generation in platelets

Thrombin-induced platelet activation leads to tyrosine phosphorylation of hematopoietic lineage cell-specific protein-1 (HS1), a 75 kDa adapter protein expressed exclusively in cells of hematopoietic lineage. We have shown HS1 to be a functionally important signaling molecule downstream of PAR-4 and GPVI collagen receptor. We have thus begun to elucidate PAR signaling pathway of HS1 phosphorylation, and its functional implications. PAR-1 and PAR-4 activating peptides (SFLLRN and AYPGKF, respectively) induced HS1 phosphorylation in a Gq-dependent manner as shown by incubation with the Gq inhibitor, YM254890. Consistently, HS1 phosphorylation was abolished in platelets from Gq deficient mice upon AYPGKF stimulation. Treatment with ADP receptor antagonists did not affect HS1 phosphorylation. Pretreatment of platelets with Src kinase inhibitors abolished HS1 phosphorylation. Further Syk activation, as measured by tyrosine phosphorylation of Syk (residues 525/526), in response to PAR activation was abolished in the presence of Src inhibitors. HS1 null mice show inhibition of PAR-mediated thromboxane A2 generation compared to wild type littermates. Phosphorylation of Erk, a key signaling molecule in thromboxane generation, was also diminished in HS1 null mice platelets. Based on these findings, we conclude that tyrosine phosphorylation of HS1 occurs downstream of both PAR-1 and PAR-4. HS1 phosphorylation is a Gq mediated response regulated by Src kinases. Thus, HS1 may mediate PAR-induced thromboxane generation through regulation of Erk phosphorylation.