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Undoped graphene is a gapless semiconductor; however, when placed under a high perpendicular magnetic field and cooled to low temperature, it develops an insulating state. This state, dubbed ν= 0, is due to the interplay between electronic interactions and the four-fold spin and valley degeneracies in the flat band formed by the n= 0 Landau level. The nature of the ground state of ν= 0, including its spin and valley polarization, is still under debate. Here we observe vanishing bulk thermal transport in monolayer at ν= 0, in contradiction with the expected ground state, which is predicted to have finite thermal conductance even at very low temperature. Our results highlight the need for further investigations on the nature of ν= 0.

Previous studies have shown that ligand or immunoaffinity chromatography can be used to purify the human platelet thromboxane A2 (TXA2) receptor-Galphaq complex. The same principle of co-elution was used to identify another G-protein associated with platelet TXA2 receptors. It was found that in addition to Galphaq, purification of TXA2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the co-purification of a member of the G12 family. Using an antipeptide antibody specific for the human G13 alpha-subunit, this G-protein was identified as Galpha13. In separate experiments, it was found that the TXA2 receptor agonist U46619 stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) incorporation into G13 alpha-subunit. Further evidence for functional coupling of G13 to TXA2 receptors was provided in studies where solubilized platelet membranes were subjected to immunoaffinity chromatography using an antibody raised against native TXA2 receptor protein. It was found that U46619 induced a significant decrease in Galphaq and Galpha13 association with the receptor protein. These results indicate that both Galphaq and Galpha13 are functionally coupled to TXA2 receptors and dissociate upon agonist activation. Furthermore, this agonist effect was specifically blocked by pretreatment with the TXA2 receptor antagonist, BM13.505. Taken collectively, these data provide direct evidence that endogenous Galpha13 is a TXA2 receptor-coupled G-protein, as: 1) its alpha-subunit can be co-purified with the receptor protein using both ligand and immunoaffinity chromatography, 2) TXA2 receptor activation stimulates GTPgammaS binding to Galpha13, and 3) Galpha13 affinity for the TXA2 receptor can be modulated by agonist-receptor activation.

Although it is well established that cAMP inhibits platelet activation induced by all agonists, the thromboxane A(2) signal transduction pathway was found to be particularly sensitive to such inhibition. Therefore, we examined whether cAMP-dependent kinase mediates phosphorylation of the thromboxane A(2) receptor-G-protein complex. It was found that cAMP induces protein kinase A-dependent [gamma-(32)P]ATP labeling of solubilized membrane proteins in the region of Galpha subunits, i.e. 38-45 kDa. Moreover, ligand affinity chromatography purification of thromboxane A(2) receptor-G-protein complexes from these membranes revealed that 38-45-kDa phosphoproteins co-purify with thromboxane A(2) receptors. Immunoprecipitation of the affinity column eluate with a Galpha(13) antibody demonstrated that 8-Br-cAMP increased phosphorylation of thromboxane A(2) receptor-associated Galpha(13) by 87 +/- 27%. In separate experiments, immunopurification of Galpha(13) on microtiter wells coated with a different Galpha(13) antibody revealed that 8-Br-cAMP increased Galpha(13) phosphorylation by 53 +/- 19%. Finally, treatment of (32)P-labeled whole platelets with prostacyclin resulted in a 90 +/- 14% increase in phosphorylated Galpha(13) that was abolished by pretreatment with the adenylate cyclase inhibitor MDL-12. These results provide the first evidence that protein kinase A mediates phosphorylation of Galpha(13) both in vitro and in vivo and provides a basis for the preferential inhibition of thromboxane A(2)-mediated signaling in platelets by cAMP.

The mechanism by which the active metabolites of arachidonic acid (AA), i.e., thromboxane A2 and/or prostaglandin H2 (TXA2/PGH2) induce platelet aggregation is not understood. Several reports have suggested that AA-stimulated aggregation is mediated by secreted ADP, whereas other studies have proposed that this response is ADP-independent. In the present report, we used the specific TXA2/PGH2 receptor antagonist, 13-azaprostanoic acid (13-APA), and the ADP antagonist, ATP, to examine the contribution of TXA2/PGH2 or secreted ADP to aggregation. We found that 13-APA, but not ATP, deaggregates platelets stimulated by AA or U46619 (a TXA2/PGH2 mimetic). In contrast, ADP-induced aggregation was reversed in response to ATP but not to 13-APA. These results suggest that TXA2/PGH2-stimulated aggregation is mediated through TXA2/PGH2 receptor occupation. Furthermore, secreted ADP does not appear to be required for maintenance of the AA-aggregation response.

The present study evaluated the direct involvement of thromboxane A2/prostaglandin H2 (TXA2/PGH2) in the process of thrombus formation at a site of vascular damage. De-endothelialization of the rabbit aorta was performed by a balloon catheter technique. Platelet deposition to the injured vessel was measured using 111Indium-labeled autologous platelets. Studies using the radiolabeled TXA2/PGH2 antagonist, 13-azaprostanoic acid (13-APA), indicated that 13-APA has an in vivo half-life of approximately 35 min and is excreted by the kidney in the metabolized form. Addition of 13-APA to rabbit plasma samples in vitro produced a dose-dependent inhibition of arachidonic acid-induced platelet aggregation. When comparable plasma levels of 13-APA were achieved by infusion of 13-APA (300 micrograms/kg/min for 90 min), a similar dose-dependency of inhibition of ex vivo aggregation was observed. Furthermore, at a plasma concentration of 40 microM, 13-APA was found to inhibit platelet deposition to the de-endothelialized rabbit aorta by 45%. Because 13-APA does not interfere with arachidonic acid metabolism, the ability of 13-APA to suppress thrombus formation is presumably due to direct antagonism of TXA2/PGH2 at the platelet receptor level. These findings, therefore, provide evidence that TXA2 and/or PGH2 have a major role in platelet deposition at a site of vascular damage.

The presence of functional thromboxane A2 receptors in neonatal rat oligodendrocytes and human oligodendroglioma cells was investigated using immunocytochemistry, ligand affinity chromatography, radioligand binding analysis, immunoblot analysis, and calcium mobilization studies. Immunocytochemical studies revealed the presence of receptor protein on both oligodendrocytes and human oligodendroglioma cells. Ligand affinity chromatography allowed for the purification of a protein with an electrophoretic mobility (55 kDa) indistinguishable from human platelet thromboxane A2 receptors. This affinity purified protein was immunoreactive against a polyclonal anti-thromboxane A2 receptor antibody. Intact human oligodendroglioma cells specifically bound [3H]SQ29,548 with a KD of 4 nM and were found to have approximately 3500 binding sites per cell. Human oligodendroglioma cells also demonstrated calcium mobilization in response to receptor activation with U46619. These results demonstrate the presence of a functional thromboxane A2 receptor in oligodendrocytes and are consistent with previous observations indicating a high density of thromboxane A2 receptors in myelinated brain and spinal cord fiber tracts.

The relative contribution of thromboxane (TX)A2/prostaglandin (PG)H2 and ADP to platelet shape change was determined using the specific TXA2/PGH2 antagonist, 13-azaprostanoic acid (13-APA) and the ADP antagonist, ATP. Shape change was induced in human platelet-rich plasma with doses of arachidonic acid (AA) or the PG endoperoxide analog U46619, which did not cause measurable ATP/ADP release. Pretreatment of platelet-rich plasma with 13-APA completely inhibited shape change to AA or U46619 but did not inhibit ADP-induced shape change. In contrast, ATP completely blocked shape change to ADP but not to AA or U46619. In addition, 13-APA and ATP also selectively reversed shape change. Thus, 13-APA added 1.5 min subsequent to AA or U46619 resulted in almost complete reversal of the shape change response. However, 13-APA did not reverse ADP-induced shape change. A similar selectivity was observed with ATP which reversed shape change to ADP but not to AA or U46619. These findings provide evidence that the interaction of TXA2/PGH2 with its receptor results in the direct stimulation of shape change independent of secreted ADP. Furthermore, maintenance of this shape change response appears to require continued occupation of the TXA2/PGH2 receptor.

The present study investigated the relationship between inositol 1,4,5-trisphosphate (IP3), thromboxane (TX)A2 and ADP in IP3-induced activation of saponin-permeabilized platelets. The time course of the different responses indicated that IP3-induced Ca++ mobilization and TXA2 production preceded both aggregation and secretion. Furthermore, platelet aggregation occurred coincident with secretion. In contrast, U46619- [15(S)-hydroxy-11,9-epoxymethano-prosta-5Z,13E-dienoic acid] and A23187-induced aggregation was commensurate with Ca++ mobilization, and preceded the secretion response. Indomethacin and SQ29,548 ([1S-[1 alpha,2 beta(5Z),3 beta,4 alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]- 7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) inhibited IP3-mediated aggregation by 100%, Ca++ mobilization by 80% and secretion by 90%. A23187 exhibited a different inhibition profile, in that only secretion was blocked (65%). U46619-induced responses were completely inhibited by SQ29,548 and unaffected by indomethacin. Pretreatment of the platelets with creatine phosphate/creatine phosphokinase (CP/CPK), which removes secreted ADP, produced 100% inhibition of IP3-induced aggregation, 90% inhibition of Ca++ mobilization and 72% inhibition of secretion. On the other hand, CP/CPK was ineffective in blocking any of the A23187-induced responses. Concerning U46619, CP/CPK produced a 30% attenuation of maximal aggregation and 78% inhibition of both Ca++ mobilization and secretion. These results in saponin-permeabilized platelets demonstrate that IP3-induced aggregation is a secretion-mediated process which requires both TXA2 and secreted ADP. Taken together, the findings suggest that IP3 is not capable of directly causing platelet aggregation, but may function in platelets to amplify an initial agonist response through TXA2 production and secretion.

Thrombin promotes the formation of arterial thrombi by converting fibrinogen to fibrin and by causing platelets to aggregate. We have examined the combined effects of plasminogen activators and inhibitors of platelet aggregation on the lysis of platelet-rich fibrin clots formed by alpha-thrombin in citrated platelet-rich plasma. The extent of platelet aggregation and clot formation were measured by recording light transmission in an aggregometer. Immediately after the formation of platelet-rich fibrin clots, addition of 2,000 U/ml streptokinase or 50 micrograms/ml recombinant tissue-type plasminogen activator alone resulted in the degradation of polymerized fibrin and the release of trapped platelet aggregates without causing significant platelet deaggregation. Preincubation of the platelet-rich plasma with 20 microM indomethacin for 1 min before thrombin stimulation or simultaneous addition of prostaglandin E1 (10 microM) with the plasminogen activators after thrombin stimulation resulted in spontaneous platelet deaggregation. Because platelet aggregation is, in part, mediated by the binding of Arg-Gly-Asp-containing adhesive proteins to activated platelets, the effect of Arg-Gly-Asp peptides on platelet deaggregation was examined. By itself, Gly-Arg-Gly-Asp-Ser-Pro specifically caused dose- and time-dependent deaggregation of platelet aggregates formed by ADP or by thrombin in the presence of 1 mM Gly-Pro-Arg-Pro, but had no effect on the dissociation of thrombin-induced platelet-rich fibrin clots. In combination with streptokinase or recombinant tissue-type plasminogen activator, Gly-Arg-Gly-Asp-Ser-Pro enhanced the rate of lysis of platelet-rich fibrin clots. The control Gly-Arg-Gly-Glu-Ser-Pro peptide was completely ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)