Phosphoinositide 3-kinase p110α negatively regulates thrombopoietin-mediated platelet activation and thrombus formation.

Phosphoinositide 3-kinase (PI3K) plays an important role in platelet function and contributes to platelet hyperreactivity induced by elevated levels of circulating peptide hormones, including thrombopoietin (TPO). Previous work established an important role for the PI3K isoform; p110ß in platelet function, however the role of p110α is still largely unexplored. Here we sought to investigate the role of p110α in TPO-mediated hyperactivity by using a conditional p110α knockout (KO) murine model in conjunction with platelet functional assays. We found that TPO-mediated enhancement of collagen-related peptide (CRP-XL)-induced platelet aggregation and adenosine triphosphate (ATP) secretion were significantly increased in p110α KO platelets. Furthermore, TPO-mediated enhancement of thrombus formation by p110α KO platelets was elevated over wild-type (WT) platelets, suggesting that p110α negatively regulates TPO-mediated priming of platelet function. The enhancements were not due to increased flow through the PI3K pathway as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) formation and phosphorylation of Akt and glycogen synthase kinase 3 (GSK3) were comparable between WT and p110α KO platelets. In contrast, extracellular responsive kinase (ERK) phosphorylation and thromboxane (TxA2) formation were significantly enhanced in p110α KO platelets, both of which were blocked by the MEK inhibitor PD184352, whereas the p38 MAPK inhibitor VX-702 and p110α inhibitor PIK-75 had no effect. Acetylsalicylic acid (ASA) blocked the enhancement of thrombus formation by TPO in both WT and p110α KO mice. Together, these results demonstrate that p110α negatively regulates TPO-mediated enhancement of platelet function by restricting ERK phosphorylation and TxA2 synthesis in a manner independent of its kinase activity.

Circulating primers enhance platelet function and induce resistance to antiplatelet therapy.

BACKGROUND: Aspirin and P2Y12 antagonists are antiplatelet compounds that are used clinically in patients with thrombosis. However, some patients are 'resistant' to antiplatelet therapy, which increases their risk of developing acute coronary syndromes. These patients often present with an underlying condition that is associated with altered levels of circulating platelet primers and platelet hyperactivity. Platelet primers cannot stimulate platelet activation, but, in combination with physiologic stimuli, significantly enhance platelet function. OBJECTIVES: To explore the role of platelet primers in resistance to antiplatelet therapy, and to evaluate whether phosphoinositide 3-kinase (PI3K) contributes to this process. METHODS AND RESULTS: We used platelet aggregation, thromboxane A2 production and ex vivo thrombus formation as functional readouts of platelet activity. Platelets were treated with the potent P2Y12 inhibitor AR-C66096, aspirin, or a combination of both, in the presence or absence of the platelet primers insulin-like growth factor-1 (IGF-1) and thrombopoietin (TPO), or the Gz-coupled receptor ligand epinephrine. We found that platelet primers largely overcame the inhibitory effects of antiplatelet compounds on platelet functional responses. IGF-1-mediated and TPO-mediated, but not epinephrine-mediated, enhancements in the presence of antiplatelet drugs were blocked by the PI3K inhibitors wortmannin and LY294002. CONCLUSIONS: These results demonstrate that platelet primers can contribute to antiplatelet resistance. Furthermore, our data demonstrate that there are PI3K-dependent and PI3K-independent mechanisms driving primer-mediated resistance to antiplatelet therapy.

Physiological effects of a fish oil supplement on captive juvenile tuatara (Sphenodon punctatus).

Tuatara (Sphenodon, Order Sphenodontia) are rare New Zealand reptiles whose conservation involves captive breeding. Wild tuatara eat seabirds, which contain high levels of the long-chain n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These fatty acids are absent from the captive diet, and consequently, plasma fatty acid composition of wild and captive tuatara differs. This study investigated the effects of incorporating EPA and DHA into the diet of captive juvenile tuatara (Sphenodon punctatus) in an attempt to replicate the plasma fatty acid composition of wild tuatara. Tuatara receiving a fish oil supplement containing EPA and DHA showed overall changes in their plasma fatty acid composition. Phospholipid EPA and DHA increased markedly, reaching 10.0% and 5.9 mol%, respectively, by 18 mo (cf.

Imidazoline compounds inhibit KATP channels in guinea pig ventricular myocytes.

Phentolamine and related imidazolines inhibit KATP channel activity in the pancreatic beta cell. In the present study, the effects of several imidazoline-based compounds were examined upon KATP channel activity in guinea pig ventricular myocytes. Phentolamine produced a potent inhibition of KATP channel activity when examined in either excised inside-out patches or in the whole-cell configuration. This effect was unrelated to phentolamine's ability to antagonise alpha-adrenoceptors since the nonselective alpha-adrenoceptor antagonists, benextramine and phenoxybenzamine, failed to affect channel activity. Furthermore, the alpha-adrenoceptor agonist clonidine together with several related imidazolines inhibited channel activity. This suggests that imidazoline compounds modulate KATP channel activity in guinea pig ventricular myocytes and this may have clinical implications for the use of such agents as hypoglycemic drugs.

Cloning and functional expression of the cDNA encoding an inwardly-rectifying potassium channel expressed in pancreatic beta-cells and in the brain.

A cDNA clone encoding an inwardly-rectifying K-channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP-sensitive K-channel rcKATP (KATP-1;[6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression in Xenopus oocytes produced currents which were K(+)-selective, time-independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside-out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP-1 suggests that BIR1 is a subunit of a brain and beta-cell KATP channel. However, pharmacological differences and the lack of ATP-sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.

Cloning and functional expression of a rat heart KATP channel.

Potassium channels that are ATP-sensitive (KATP) couple membrane potential to the metabolic status of the cell. KATP channels are inhibited by intracellular ATP and are stimulated by intracellular nucleotide diphosphates. KATP channels are important regulators of secretory processes and muscle contraction, and are targets for therapeutic treatment of type II diabetes by the inhibitory sulphonylureas and for hypertension by activators such as pinacidil. In cardiac tissue, KATP channels are central regulators of post-ischaemic cardioprotection. Electrophysiological and pharmacological characteristics vary among KATP channels recorded from diverse tissues suggesting extensive molecular heterogeneity. A complementary DNA encoding a KATP channel was isolated from rat heart using the polymerase chain reaction. We report here that the expressed channels possess all of the essential features of native cardiac KATP channels, including sensitivity to intracellular nucleotides. In addition the cloned channels are activated by the potassium channel opener, pinacidil, but are not inhibited by the sulphonylurea, glibenclamide.

Molecular biology of the voltage-gated potassium channels of the cardiovascular system.

K+ channels represent the most diverse class of voltage-gated ion channels in terms of function and structure. Voltage-gated K+ channels in the heart establish the resting membrane K+ permeability, modulate the frequency and duration of action potentials, and are targets of several antiarrhythmic drugs. Consequently, an understanding of K+ channel structure-function relationships and pharmacology is of great practical interest. However, the presence of multiple overlapping currents in native cardiac myocytes complicates the study of basic K+ channel function and drug-channel interactions in these cells. The application of molecular cloning technology to cardiovascular K+ channels has identified the primary structure of these proteins, and heterologous expression systems have allowed a detailed analysis of channel function and pharmacology without contaminating currents. To date six different K+ channels have been cloned from rat and human heart, and all have been functionally characterized in either Xenopus oocytes or mammalian tissue culture systems. This initial research is an important step toward understanding the molecular basis of the action potential in the heart. An important challenge for the future is to determine the cell-specific expression and relative contribution of these cloned channels to cardiac excitability.

Functional characterization of RK5, a voltage-gated K+ channel cloned from the rat cardiovascular system.

A voltage-sensitive K+ channel previously cloned from rat heart designated RK5 (rat Kv4.2) (Roberds and Tamkun, 1991, Proc. Natl. Acad. Sci. USA 88, 1798-1802) was functionally characterized in the Xenopus oocyte expression system. RK5 is a homolog of the Drosophila Shal K+ channel, activates with a rise time of 2.8 ms, has a midpoint for activation of -1 mV and rapidly inactivates with time constants of 15 and 60 ms. RK5 is sensitive to 4-AP, IC50 = 5 mM, and is insensitive to TEA and dendrotoxins. The voltage dependence and kinetics of the RK5 induced currents suggest this channel contributes to the Ito current in heart.

Development of pharmacological sensitivity to adenosine analogs in embryonic chick heart: role of A1 adenosine receptors and adenylyl cyclase inhibition.

The developing chick heart was employed as a model system to explore temporal correlations between the onset of pharmacological sensitivity to adenosine analogs and the appearance of A1 adenosine receptors coupled to adenylyl cyclase. A characterization of the developmental profile for adenosine analog-induced negative chronotropic response revealed that isolated atria from 5- and 6-day embryos were unresponsive to adenosine analogs. The onset of pharmacological sensitivity occurred on embryonic day 7, as evidenced by a 27% reduction in atrial beating rate in the presence of 2-chloradenosine (2-CIA) (30 microM). The sensitivity of embryonic atria to 2-CIA increased continuously from day 7 to day 12 in ovo, when the atria became fully responsive to the negative chronotropic effect of this adenosine analog. In order to evaluate whether the developmental increase in pharmacological sensitivity to 2-CIA reflected changes in the number of A1 adenosine receptors, the ontogenesis of A1 adenosine receptors was assessed using the antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine as a probe. Cardiac membranes from day 5 and day 6 embryos possessed approximately one third of the maximum number of A1 adenosine receptors expressed at later embryonic ages. Additionally, agonist/[3H] DPCPX competition curves revealed that the high affinity state receptors comprised a larger proportion of the total receptor population in membranes from day 6 as compared with day 12 embryos. These results suggest that there are pharmacologically inactive A1 receptors in hearts from day 5 and day 6 embryos. The developmental change in A1 receptor-mediated negative chronotropic response paralleled the increase in [3H]DPCPX binding sites from embryonic day 7 to day 10. Thus, a large fractional occupancy of A1 adenosine receptors is required to express negative chronotropy during this period of embryonic development. Studies of the sensitivity of adenylyl cyclase to inhibition by cyclopentyladenosine as a function of ontogenesis revealed that cyclopentyladenosine inhibited basal adenylyl cyclase activity to a similar maximal extent from embryonic day 5 through day 16. The efficacy of cyclopentyladenosine as an inhibitor of adenylyl cyclase activity was, therefore, stable during a developmental period when A1 receptor density increased approximately 3-fold. Hence, only a fraction of the A1 receptors present during embryogenesis need to be coupled to produce a maximum response with respect to adenylyl cyclase inhibition, which is an indication of the presence of spare receptors. receptors.(ABSTRACT TRUNCATED AT 400 WORDS)