Jianguo Jin

Molecular Basis for ADP-induced Platelet Activation II. THE P2Y1 RECEPTOR MEDIATES ADP-INDUCED INTRACELLULAR CALCIUM MOBILIZATION AND SHAPE CHANGE IN PLATELETS

ADP is an important platelet agonist causing shape change from smooth discoid shape to spiculated spheres and platelet aggregation. However, the molecular mechanisms involved in ADP-induced platelet activation have not been elucidated. We demonstrated earlier the existence of two distinct ADP receptors on platelets, one coupled to phospholipase C, P2TPLC, and the other to inhibition of adenylyl cyclase, P2TAC (Daniel, J. L., Dangelmaier, C., Jin, J., Ashby, B., Smith, J. B., and Kunapuli, S. P. (1998)J. Biol. Chem. 273, 2024–2029), in addition to the previously described P2X1 receptor. Here we report the cloning of a cDNA clone encoding the P2Y1 receptor from a human platelet cDNA library by homology screening with radiolabeled P2Y1-P2Y6 receptor cDNAs. ADP or 2-methyl(thio)-ADP-induced intracellular calcium increases were inhibited by the P2Y1 receptor-specific antagonists, adenosine 3′-phosphate 5′-phosphosulfate (A3P5PS), adenosine 3′-phosphate 5′-phosphate (A3P5P), and adenosine 2′-phosphate 5′-phosphate (A2P5P), in a concentration-dependent manner, but not by ARL 66096 or α,β-MeATP. A3P5PS, A3P5P, and A2P5P also inhibited the shape change of aspirinated platelets induced by 10 μm ADP or 3 μm 2-methyl(thio)-ADP in a concentration-dependent manner, with complete inhibition occurring at 300 μm. On the other hand ARL 66096 (100 nm), a potent P2TAC antagonist and α,β-methylene-ATP (40 μm), a P2X1 receptor agonist, had no effect on ADP-induced platelet shape change. On the contrary, ADP-induced inhibition of adenylyl cyclase was blocked by ARL 66096, but not by α,β-MeATP or the P2Y1 receptor-specific antagonists, A3P5PS, A3P5P, or A2P5P. These results demonstrate the role of the P2Y1 receptor in ADP-induced platelet shape change and calcium mobilization and support the idea that several P2 receptors are involved in the regulation of different aspects of platelet stimulus-response coupling.

Molecular basis for ADP-induced platelet activation, I : Evidence for three distinct ADP receptors on human platelets

Acting through cell surface receptors, ADP activates platelets resulting in shape change, aggregation, thromboxane A2 production, and release of granule contents. ADP also causes a number of intracellular events including inhibition of adenylyl cyclase, mobilization of calcium from intracellular stores, and rapid calcium influx in platelets. However, the receptors that transduce these events remain unidentified and their molecular mechanisms of action have not been elucidated. The receptor responsible for the actions of ADP on platelets has been designated the P2T receptor. In this study we have used ARL 66096, a potent antagonist of ADP-induced platelet aggregation, and a P2X ionotropic receptor agonist, α,β-methylene adenosine 5′-triphosphate, to distinguish the ADP-induced intracellular events. ARL 66096 blocked ADP-induced inhibition of adenylyl cyclase, but did not affect ADP-mediated intracellular calcium increases or shape change. Both ADP and 2-methylthio-ADP caused a 3-fold increase in the level of inositol 1,4,5-trisphosphate over control levels which peaked in a similar fashion to the Ca2+ transient. The increase in inositol 1,3,4-trisphosphate was of similar magnitude to that of inositol 1,4,5-trisphosphate. α,β-Methylene adenosine 5′-triphosphate did not cause an increase in either of the inositol trisphosphates. These results clearly demonstrate the presence of two distinct platelet ADP receptors in addition to the P2X receptor: one coupled to adenylyl cyclase and the other coupled to mobilization of calcium from intracellular stores through inositol trisphosphates.

Molecular Mechanism of Thromboxane A2-induced Platelet Aggregation ESSENTIAL ROLE FOR P2T AC and α2ARECEPTORS

Thromboxane A2 is a positive feedback lipid mediator produced following platelet activation. The Gq-coupled thromboxane A2 receptor subtype, TPα, and Gi-coupled TPβ subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T AC , coupled to Gq and Gi, respectively. We investigated whether the stable thromboxane A2 mimetic, (15S)-hydroxy-9,11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through Gq and Gi, through co-activation of TPα and TPβ receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T AC receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an α2A-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the Gi pathway by selective activation of either the P2T AC receptor or the α2A-adrenergic receptor. We conclude that whereas thromboxane A2 causes intracellular calcium mobilization and shape change independently, thromboxane A2-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate Gi-coupled receptors.

Distribution of P2Y receptor subtypes on haematopoietic cells

1 RT–PCR‐southern hybridization analyses with radiolabelled P2Y receptor cDNAs as probes indicated that the peripheral blood leukocytes and the human umbilical vein endothelial cells express P2Y1, P2Y2, P2Y4 and P2Y6 receptors. 2 Of the haematopoietic cell lines tested, promonocytic U937 cells express P2Y2 and P2Y6, but not P2Y1 or P2Y4; promyelocytic HL‐60 cells express the P2Y1, P2Y2 and P2Y6 receptors but not the P2Y4 receptor; K562 cells express P2Y1 but not P2Y2, P2Y4 or P2Y6; and Dami cells express P2Y1, P2Y2, P2Y4 and P2Y6 receptors. 3 Of the peripheral blood leukocytes tested, polymorphonuclear cells express P2Y4 and P2Y6 but not P2Y1 or P2Y2 receptors; monocytes express P2Y1, P2Y2, P2Y4 and P2Y6 receptors and lymphocytes express P2Y1, P2Y2, P2Y4 and P2Y6 receptors. 4 These results suggest a physiological role for different P2Y receptor subtypes in the extracellular nucleotide‐mediated stimulation of monocytes, neutrophils, lymphocytes and endothelial cells.

Cardiotoxic and Cardioprotective Features of Chronic β-Adrenergic Signaling

Rationale: In the failing heart, persistent &bgr;-adrenergic receptor activation is thought to induce myocyte death by protein kinase A (PKA)-dependent and PKA-independent activation of calcium/calmodulin-dependent kinase II. &bgr;-adrenergic signaling pathways also are capable of activating cardioprotective mechanisms. Objective: This study used a novel PKA inhibitor peptide to inhibit PKA activity to test the hypothesis that &bgr;-adrenergic receptor signaling causes cell death through PKA-dependent pathways and cardioprotection through PKA-independent pathways. Methods and Results: In PKA inhibitor peptide transgenic mice, chronic isoproterenol failed to induce cardiac hypertrophy, fibrosis, and myocyte apoptosis, and decreased cardiac function. In cultured adult feline ventricular myocytes, PKA inhibition protected myocytes from death induced by &bgr;1-adrenergic receptor agonists by preventing cytosolic and sarcoplasmic reticulum Ca2+ overload and calcium/calmodulin-dependent kinase II activation. PKA inhibition revealed a cardioprotective role of &bgr;-adrenergic signaling via cAMP/exchange protein directly activated by cAMP/Rap1/Rac/extracellular signal-regulated kinase pathway. Selective PKA inhibition causes protection in the heart after myocardial infarction that was superior to &bgr;-blocker therapy. Conclusions: These results suggest that selective block of PKA could be a novel heart failure therapy.

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.

Constitutive activity of human prostaglandin E receptor EP3 isoforms

The human EP3 prostaglandin receptor is a seven transmembrane, G protein‐coupled receptor that couples to inhibition of adenylyl cyclase. The receptor occurs as at least six isoforms which result from alternative splicing. The isoforms are identical over the first 359 amino acids, comprising the seven transmembrane helices, but differ in the carboxyl terminal tail which ranges in length from 6 to 65 amino acids beyond the common region. We have stably expressed in CHO‐K1 cells four of the isoforms (EP3I‐EP3IV) and a form of the EP3 receptor (T‐359) truncated at the carboxyl‐terminal region defined by the alternative splicing site at amino acid number 359. Isoforms EP3I and EP3II showed concentration‐dependent inhibition of forskolin‐stimulated adenylyl cyclase in CHO‐K1 cells by the EP3 receptor agonist, sulprostone. The IC50 calculated for sulprostone inhibition was 0.2 nM for EP3I and 0.15 nM for EP3II. The maximum extent of inhibition was 80% for both isoforms. Isoforms EP3III and EP3IV showed marked constitutive activity, inhibiting forskolin‐stimulated adenylyl cyclase in the absence of agonist. EP3IV also displayed some agonist‐dependent inhibition whereas EP3III was fully constitutively active. The truncated receptor T‐359 was fully constitutively active, inhibiting forskolin‐stimulated adenylyl cyclase by about 70% in the absence of agonist, and showed no agonist‐dependent inhibition, in agreement with a similar truncation of the mouse EP3 receptor. To confirm that differences in cyclic AMP level between isoforms represent constitutive activity, we treated cells with pertussis toxin for 6 h to abolish Gi function. Pertussis toxin reversed sulprostone‐mediated inhibition of cyclic AMP formation in EP3I and EP3II and abolished constitutive activity of EP3III, EP3IV and T‐359 so that the level of forskolin‐stimulated cyclic AMP produced was the same in all cells and similar to that obtained in mock‐transfected cells. In mock‐transfected cells, sulprostone had no effect on forskolin‐stimulated cyclic AMP formation. For these experiments we chose clones that showed similar expression levels of each isoform, as determined by binding of [3H]‐prostaglandin E2 (PGE2) (EP3I, 0.71; EP3II, 1.47; EP3IV, 1.59 pmol mg−1 protein). Mock‐transfected cells showed no detectable binding of [3H]‐PGE2. In addition, we performed a detailed study of the effects of expression level on constitutive activity. Over a six fold range of expression there was no change in the properties of each isoform with regard to whether it was constitutively active or not. The degree of constitutive activity correlated with the inverse of the length of the C‐terminal tail of the isoforms. However, no correlation was found between isoforms from human and mouse: whereas EP3II shows no constitutive activity, its mouse homologue, EP3γ, shows almost complete constitutive activity, even though the C‐terminal domains of the receptors following the splice site differ in only 7 of 29 amino acids.

The C6-2B glioma cell P2YAC receptor is pharmacologically and molecularly identical to the platelet P2Y12 receptor

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6‐2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2YAC, (P2TAC or P2T in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y12 in keeping with current P2 receptor nomenclature. Three selective P2T receptor antagonists, with a range of affinities, inhibited ADP‐induced aggregation of washed human or rat platelets, in a concentration‐dependent manner, with a rank order of antagonist potency (pIC50, human: rat) of AR‐C78511 (8.5 : 9.1)>AR‐C69581 (6.2 : 6.0)>AR‐C70300 (5.4 : 5.1). However, these compounds had no effect on ADP‐induced platelet shape change. All three antagonists had no significant effect on the ADP‐induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y1 receptor, when used at concentrations that inhibit platelet aggregation. These antagonists also blocked ADP‐induced inhibition of adenylyl cyclase in rat platelets and C6‐2B cells with identical rank orders of potency and overlapping concentration – response curves. RT – PCR and nucleotide sequence analyses revealed that the C6‐2B cells express the P2Y12 mRNA. These data demonstrate that the P2YAC receptor in C6‐2B cells is pharmacologically identical to the P2TAC receptor in rat 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 phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in platelets

Summary. Background:  Myosin IIA is an essential platelet contractile protein that is regulated by phosphorylation of its regulatory light chain (MLC) on residues (Thr)18 and (Ser)19 via the myosin light chain kinase (MLCK).