Heat Equilibration of Integer and Fractional Quantum Hall Edge Modes in Graphene.

Hole-conjugate states of the fractional quantum Hall effect host counterpropagating edge channels which are thought to exchange charge and energy. These exchanges have been the subject of extensive theoretical and experimental works; in particular, it is yet unclear if the presence of integer quantum Hall edge channels stemming from fully filled Landau levels affects heat equilibration along the edge. In this Letter, we present heat transport measurements in quantum Hall states of graphene demonstrating that the integer channels can strongly equilibrate with the fractional ones, leading to markedly different regimes of quantized heat transport that depend on edge electrostatics. Our results allow for a better comprehension of the complex edge physics in the fractional quantum Hall regime.

[Comparison of British and French expatriate doctors' characteristics and motivations]. / Comparaison des caractéristiques et des motivations des médecins expatriés français et britanniques.

BACKGROUND: Migration of medical practitioners is rarely studied despite its importance in medical demography: the objective of this study was to analyze the characteristics and motivations of the French doctors settled in the United Kingdom and of the British doctors settled in France. METHODS: This cross-sectional study was conducted using a self-completed questionnaire sent to all French doctors practicing in the United Kingdom (in 2005) and all British medicine doctors practicing in France (in 2009). The doctors were identified with official data from the National Medical Councils: 244 French doctors practicing in the United Kingdom and 86 British doctors practicing in France. The questionnaire was specifically developed to determine the reasons of moving to the other country, and the level of satisfaction after expatriation. RESULTS: A total of 98 French doctors (out of 244) and 40 British doctors (out of 86) returned the questionnaire. Respondents were mainly general practitioners with a professional experience of 8 to 9 years. The sex ratio was near 1 for both groups with a majority of women among physicians under 50 years. The motivations were different between groups: French doctors were attracted by the conditions offered at the National Health Service, whereas British doctors were more interested in opportunities for career advancement, joining husband or wife, or favourable environmental conditions. Overall, the respondents considered expatriation as satisfactory: 84% of French doctors, compared with only 58% of British doctors, were satisfied with their new professional situation. CONCLUSION: This study, the first in its kind, leads to a clearer understanding of the migration of doctors between France and the United Kingdom.

Identification and functional characterization of thromboxane A2 receptors in Schwann cells.

Previous reports have demonstrated the presence of functional thromboxane A2 (TP) receptors in astrocytes and oligodendrocytes. In these experiments, the presence and function of TP receptors in primary rat Schwann cells (rSC) and a neurofibrosarcoma-derived human Schwann cell line (T265) was investigated. Immunocytochemical and immunoblot analyses using polyclonal anti-TP receptor antibodies demonstrate that both cell types express TP receptors. Treatment with the stable thromboxane A2 mimetic U46619 (10 microM) did not stimulate intracellular calcium mobilization in rSC, whereas T265 cells demonstrated a calcium response that was inhibited by prior treatment with TP receptor antagonists. U46619 also stimulated CREB phosphorylation on Ser133 in T265 cells and, to a lesser extent, in rSC. To identify potential mechanisms of CREB phosphorylation in rSC, we monitored intracellular cAMP levels following U46619 stimulation. Elevated levels of cAMP were detected in both rSC (20-fold) and T265 (15-fold) cells. These results demonstrate that TP receptor activation specifically stimulates CREB phosphorylation in T265 cells, possibly by a calcium- and/or cAMP-dependent mechanism. In contrast, TP receptor activation in rSC stimulates increases in cAMP and CREB phosphorylation but does not elicit changes in intracellular calcium.

Shifts in the affinity distribution of one class of seven-transmembrane receptors by activation of a separate class of seven-transmembrane receptors.

We have demonstrated previously that activation of thrombin receptors causes increased Galpha(q) coupling to thromboxane A(2) receptors and increased thromboxane A(2) receptor ligand affinity. These results led to the hypothesis that thrombin receptor activation stimulates Galpha(q) redistribution to thromboxane A(2) receptors, thereby shifting them to a higher affinity state. The present study investigated three questions regarding this inter-receptor signaling phenomenon: (i) does activation of thrombin receptors cause a redistribution of thromboxane A(2) receptor subpopulations; (ii) does inter-receptor signaling require that participating receptors couple to the same family of G-protein alpha-subunits; and (iii) does inter-receptor signaling occur in cell types other than platelets? It was found that thrombin receptor activation caused a shift in the thromboxane A(2) receptor binding data from a one-site model to a two-site model (K(i) = 0.5 microM vs K(i) = 10 nM and 1.1 microM for the antagonist 4-[2-[[(4-chlorophenyl)sulfonyl]amino]ethyl]benzeneacetic acid (BM13. 505) and K(i) = 2.5 microM vs K(i) = 29.5 nM and 2.6 microM for the agonist 9,11-dideoxy-9alpha,11alpha-methanoepoxy prostaglandin F(2alpha) (U46619). It also was found that activation of prostaglandin D(2) receptors also caused a shift of prostacyclin receptor binding data from a one-site model (IC(50) = 10.1 nM) to a two-site model (IC(50) = 3.3 and 12.5 nM). The physiological manifestation of this inter-receptor signaling between prostacyclin and prostaglandin D(2) receptors was a synergistic inhibition of human platelet aggregation. Finally, the present results established that activation of endothelial cell thrombin receptors shifts thromboxane A(2) receptor affinity from K(i) = 0.8 microM (control) to K(i) = 0.2 microM (thrombin receptor-activating peptide), indicating that cells other than platelets have the capability to signal between seven-transmembrane receptors.

Synthesis and biological characterization of sqbazide, a novel biotinylated photoaffinity probe for the study of the human platelet thromboxane A2 receptor.

SQBAzide, a biotinylated, azido derivative of the TXA2 receptor antagonist, SQ31,491, was synthesized and characterized. The compound specifically inhibited human platelet aggregation mediated by TXA2 receptor activation and irreversibly labeled platelet TXA2 receptors upon exposure to ultraviolet light. This probe should prove to be of significant value for the study of the receptor-ligand binding domain.

Cyclic AMP-dependent phosphorylation of thromboxane A(2) receptor-associated Galpha(13).

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.

Identification of Galpha13 as one of the G-proteins that couple to human platelet thromboxane A2 receptors.

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.

A molecular mechanism for signaling between seven-transmembrane receptors: evidence for a redistribution of G proteins.

Although activation of one seven-transmembrane receptor can influence the response of a separate seven-transmembrane receptor, e. g., the phenomenon of synergism, the underlying mechanism(s) for this signaling process is unclear. The present study investigated communication between two receptors that exhibit classical synergism, e.g., human platelet thrombin and thromboxane A2 receptors. Activation of thrombin receptors caused an increase in ligand affinity of thromboxane A2 receptors. This effect (i) was shown to be specific, since a similar increase in ligand affinity was not caused by ADP or A23187; (ii) did not require cytosolic components, e.g., kinases, proteases, phosphatases, etc., because it occurred in isolated platelet membranes; (iii) was G protein-mediated because it was blocked by an Galphaq C terminus antibody; and (iv) was associated with a net increase in Galphaq coupling to thromboxane A2 receptors. Collectively, these data provide evidence that seven-transmembrane receptors that share a common Galpha subunit can communicate with each other via a redistribution of their G proteins. Thus, activation of thrombin receptors increases Galphaq association with thromboxane A2 receptors thereby shifting them to a higher affinity state. This signaling phenomenon, which modulates receptor-ligand affinity, may serve as a molecular mechanism for cellular adaptive processes such as synergism.

The identification and characterization of oligodendrocyte thromboxane A2 receptors.

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.

Labeling of human platelet plasma membrane thromboxane A2/prostaglandin H2 receptors using SQB, a novel biotinylated receptor probe.

This study reports the synthesis, biological evaluation, and application of a new biotinylated derivative 1-[[1S-[1 alpha, 2 alpha (Z),3 alpha, 4 alpha]]-7-[3-[[[[(1-oxocyclohexylpropyl)amino]acetyl]amino] methyl]-7-oxabicyclo [2.2.1]hept-2-yl]-5-heptenoyl]-2-[hexahydro-2'-oxo-1H-thieno[3',4' d] imidazole-4'-pentanoyl]hydrazine (SQB) of the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor antagonist [1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-7-[3-[[[[(1-oxocyclohexylpropyl)amino]acetyl] amino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ31,491). SQB was synthesized by reacting SQ31,491 with biotin hydrazide, and the product was purified by flash chromatography. It was found that SQB specifically inhibited platelet aggregation in response to U46619 with an IC50 of 275 nM. On the other hand, SQB did not inhibit adenosine diphosphate or A23187-induced aggregation. Competition binding studies revealed that SQB produced a concentration-dependent inhibition of [3H]-[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 ([3H]SQ29,548) specific binding in 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS)-solubilized platelet membranes, with a Ki of 220 nM. The shape of the SQB inhibition binding curve was indistinguishable from that produced by the TXA2/PGH2 receptor antagonist BM13.177. Finally, incubation of gel-filtered platelets or platelet-rich plasma with SQB and fluorescein isothiocyanate (FITC)-avidin demonstrated fluorescent labeling of platelet plasma membrane TXA2/PGH2 receptors. Furthermore, this SQB-FITC fluorescent labeling was reduced significantly by co-incubation of the platelets with the TXA2/PGH2 antagonist SQ29,548. Based on the ability of SQB-FITC-avidin to label intact platelets, it can be concluded: (1) that a pool of platelet TXA2/PGH2 receptors resides in the plasma membrane; and (2) that the binding domains for these receptors are oriented at or near the external membrane surface. Collectively, these data demonstrate that SQB is a highly specific probe for TXA2/PGH2 receptors, which should be of significant value for receptor localization studies in platelets and other tissues.

Thromboxane A2 mediates the stimulation of inositol 1,4,5-trisphosphate production and intracellular calcium mobilization by bradykinin in neonatal rat ventricular cardiomyocytes.

Bradykinin is a mediator of the protection of myocardium by angiotensin I-converting enzyme/kininase II inhibitors. We reported that the activation of B2 bradykinin receptors in neonatal rat cardiac myocytes in primary culture was followed by hydrolysis of phosphatidylinositol 4,5-bisphosphate and formation of inositol 1,4,5-trisphosphate (IP3). Here we examine the regulation of IP3 formation stimulated by bradykinin. Activation of myocytes with 1 mu/L bradykinin increased IP3 production from 117 +/- 8.3 to 1011 +/- 48.6 pmol/mg protein. Treatment of the cells with 10 mu/L indomethacin or 1 mu/L dexamethasone partially blocked this bradykinin-induced response. Moreover, either U73122, a phospholipase C inhibitor, or (p-amylcinnamoyl) anthranilic acid, a phospholipase A2 inhibitor, blunted the IP3 response to bradykinin. Because thromboxane A2 stimulates inositol bisphosphate metabolism in guinea pig atria, we also investigated the effect of the thromboxane A2 receptor antagonist BM 13177 (1 mu/L), which strongly attenuated the stimulated IP3 production. Since thromboxane A2 appears to partly mediate the IP3 response to bradykinin, we examined the effect of the stable thromboxane A2 mimetic U46619. Control cultures were stimulated more by U46619 than by bradykinin (1629 +/- 14.5 versus 1011 +/- 48.6 pmol IP3/mg protein). This property of U46619 was selectively antagonized by BM 13177. Inhibition of either phospholipase C or phospholipase A2 blunted the IP3 response to U46619. Short-term (30 minutes) activation of protein kinase C with phorbol 12-myristate 13-acetate (10 pmol/L to 1 mu/L) attenuated the IP3 accumulation in response to bradykinin; the effect of phorbol 12-myristate 13-acetate was reversed with 1 mu/L staurosporine, a protein kinase C inhibitor. Treatment with 1 microgram/mL cholera toxin or pertussis toxin for 4 hours amplified the IP3 response to 10 nmol/L bradykinin from 570 +/- 20.0 to 1150 +/- 51.3 and to 1016.7 +/- 21.9 pmol/mg protein. Bradykinin mobilized 9.4% of intracellular calcium stores in cardiomyocytes as assessed by chlortetracycline-based fluorometry, and this effect of bradykinin was blocked by BM 13177 or the B2 bradykinin receptor blocker Hoe 140 by more than 70%. In functional studies, bradykinin (1 mu/L) increased by 12% the twitch contractile force of neonatal rat ventricular strips paced at threshold intensity, but this was unaffected by BM 13177. In conclusion, in cardiomyocytes, bradykinin enhances IP3 production mostly via phospholipase A2 stimulation and thromboxane A2 formation. This prostanoid in turn stimulates its receptor and activates phospholipase C, which then splits phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglycerol. The effect of bradykinin on phospholipase C, via thromboxane A2, is negatively regulated by protein kinase C activation.

Localization of authentic thromboxane A2/prostaglandin H2 receptor in the rat kidney.

Using a polyclonal antibody against authentic thromboxane A2/prostaglandin H2 (TxA2/PGH2) receptor protein, we assessed the distribution of this receptor in the normal rat kidney by routine methods of immunofluorescence microscopy. The receptor localized both in glomeruli and in tubules. In the former, the distribution of the receptor was most prominent along the lumen of glomerular capillary loops. Parietal epithelial cells of the Bowman's capsule, podocytes and mesangial cells also demonstrated immunostainable receptor. In the tubules, the receptor localized most prominently at the base of the brush border of proximal tubules and at the luminal surface of thick ascending limbs and distal convoluted tubules. These observations point to sites that are likely to be targeted by thromboxane A2 in forms of renal injury characterized by enhanced synthesis of this eicosanoid.

Nonspecific protease-catalyzed hydrolysis/synthesis of a mixture of peptides: product diversity and ligand amplification by a molecular trap.

We sought to develop a peptide library in solution and dynamically screen this library for peptides that would bind to macromolecules of interest. Peptide diversity was achieved in an initial stock solution of peptides by using proteases under conditions in which both hydrolysis and synthesis occurred. As an example, a simple reaction containing YGG, FL and thermolysin resulted in the synthesis of YGGFL as well as many other undefined products. When low molecular weight products of a reaction containing VA, AL, and thermolysin were subsequently exposed to dipeptidase, 7 out of 9 potential dipeptides were observed. Incubation of protease with an hydrolysate of albumin and a radiolabeled peptide resulted in the radiolabel participating in reactions other than simple hydrolysis and, after 24 h, the specific activity of radiolabel was shown by high performance liquid chromatography to disperse to a level that would be necessary in the event of maximum theoretical diversity. When a binding macromolecule was exposed to this system, ligand production was amplified relative to reactions run in the absence of binding macromolecule. This protease-based peptide scrambling and binding system was utilized for the discovery of novel peptides that bind to fibrinogen.

Endothelin-3 reduces Ca(2+)-uptake and Ca2+ content of platelet internal stores.

In stimulated platelets, endothelin-3 (ET-3) has been previously shown to attenuate Ca2+ mobilization. Using the calcium indicator chlortetracycline, the present study demonstrates that 0.5 microM ET-3 produced a 24% reduction in the Ca2+ pool mobilized by A23187. ET-3 up to 1 microM dose-dependently decreased the initial velocity and steady state value of 45Ca(2+)-uptake into platelet membrane vesicules (p < 0.001). In addition, ET-3 partially reversed the inhibitory effects of half maximally effective concentrations of thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone, two specific inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPases. These results suggest that ET-3 is functionally coupled to Ca(2+)-pumps of the dense tubular system. Based on these findings, we propose that ET-3 decreases the activity of Ca(2+)-pumps in the dense tubular system which accumulates less Ca2+, leading to lowered Ca2+ release in response to agonists.

Endothelin-3 decreases Ca2+ uptake in platelet membrane vesicles.

To analyze the mechanisms by which endothelin-3 (ET-3) attenuates agonist-mediated Ca2+ mobilization from an internal pool, we investigated ET-3 effects on 45Ca2+ uptake in platelet membrane vesicles. They were compared to those of thapsigargin (Tg), a specific inhibitor of the dense tubule Ca2+ pumps. In the absence of ATP, ET-3 up to 1 microM did not affect the amount of Ca2+ bound to membrane sites. In the presence of ATP, ET-3 dose-dependently reduced the initial rate and the extent of Ca2+ uptake (p < 0.001). In comparison, Tg dose-dependently inhibited both the ATP-independent Ca2+ binding (p < 0.001) and the ATP-dependent Ca2+ accumulation (p < 0.001), with half-maximal effects at 7 nM. Pretreatment with 1 microM ET-3 decreased the inhibitory effect of 10 nM Tg, but only on the initial rate of ATP-dependent Ca2+ uptake (p = 0.04; n = 6). These results indicate that ET-3 is functionally coupled to Ca(2+)-ATPases of the dense tubules. Its inhibitory effects are probably due to inhibition of the catalytic cycle of the Ca2+ pumps. Such inhibition could lead to a depletion of Ca2+ stores and therefore to reduced Ca2+ release in response to agonists.

Purification of rat brain, rabbit aorta, and human platelet thromboxane A2/prostaglandin H2 receptors by immunoaffinity chromatography employing anti-peptide and anti-receptor antibodies.

In the present study, a new polyclonal antibody (TxAb) was raised against native thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor protein. Previously developed anti-peptide antibodies (P1Ab, P2Ab) and TxAb were then used to prepare immunoaffinity columns to purify TXA2/PGH2 receptors from platelets, brain, and aorta. In platelets, SDS-polyacrylamide gel electrophoresis revealed the purification of a 55-kDa protein by each affinity column. Identification of this protein as the TXA2/PGH2 receptor was based on: 1) an identical electrophoretic mobility to authentic receptor; 2) immunoblotting of TxAb against P1Ab and P2Ab-purified protein; 3) immunoblotting of P1Ab/P2Ab against TxAb-purified protein; and 4) specific [3H]SQ29,548 binding to TxAb-purified protein. P1Ab/TxAb purification of receptors from brain revealed a major protein band at 55 kDa. Furthermore, the eluates from ligand affinity chromatography confirmed the presence of this 55-kDa protein in brain (which was immunoblotted with TxAb), and contained specific [3H]SQ29,548 binding. In addition to the 55-kDa protein, P1Ab/TxAb also purified a minor protein in brain at 52 kDa, which when concentrated, cross-blotted with TxAb and P1Ab. This finding indicates sequence homology between the 55- and 52-kDa proteins. Independent identification of brain TXA2/PGH2 receptors was provided by P2Ab/TxAb immunohistochemistry, which demonstrated specific labeling of discrete myelin-containing fiber tracts. P2Ab/TxAb purification of TXA2/PGH2 receptors from aorta also revealed a major protein band at 55 kDa and a minor band at 52 kDa. These results represent the first purification of TXA2/PGH2 receptors from either brain or aorta.

Identification of Gq as one of the G-proteins which copurify with human platelet thromboxane A2/prostaglandin H2 receptors.

The present study employed ligand affinity and immunoaffinity chromatography to isolate human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor-coupled G-proteins. Purification of TXA2/PGH2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the elution of receptor binding and GTPase activity in the same fraction. GTPase activity of this fraction was enriched (6-fold) relative to solubilized platelet membranes, was stimulated (65%) by 500 nM U46619, and was blocked (74%) by 250 nM SQ29,548. Furthermore, GTP (100 microM) increased [3H]SQ29,548 receptor binding by 48%. Immunoblotting of this fraction against QL antiserum identified a 42-kDa protein as a member of the Gq family. In separate experiments, TXA2/PGH2 receptors were purified by immunoaffinity chromatography using P1Ab, P2Ab, and TxAb affinity columns. QL-immunoreactive proteins at 42 kDa were found in all three column eluates. Studies using G alpha,common antiserum (GA/1) demonstrated immunoblotting of two proteins of approximately 42 and 85 kDa in both the ligand and P2Ab affinity column fractions. On the other hand, the P1Ab and TxAb affinity column eluates contained GA/1 immunoreactivity only in the 42 kDa region. Collectively, these data identify Gq as a TXA2/PGH2 receptor-coupled G-protein and suggest the association of this receptor with additional G alpha subunits.

Anti-peptide antibodies against the human blood platelet thromboxane A2/prostaglandin H2 receptor. Production, purification and characterization.

Two anti-peptide antibodies have been raised against the human blood platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. Based on the published sequence of the placental TXA2/PGH2 receptor, two decapeptide segments were selected as potential antigens: one in the first extracellular loop corresponding to residue 89 through 98, and the other in the C-terminal region of the intracellular domain corresponding to residue 314 through 323. Rabbits were immunized with each peptide, and the antisera were subjected to a two-step purification procedure. The IgG fraction was purified using a DEAE Affi-Gel Blue column, and the peptide-specific IgG was further purified by affinity chromatography employing each peptide as the immobilized ligand. The combined purification factor for both procedures was approximately 60-fold. By ELISA, both antibodies displayed immunoreactivity toward their synthetic antigens, solubilized platelet membranes and affinity-purified TXA2/PGH2 receptor protein. Furthermore, Western blot analysis revealed that: (1) each antibody reacted with the purified platelet TXA2/PGH2 receptor protein (55 kDa); and (2) each antibody recognized a single band (55 kDa) in solubilized platelet membranes. These findings establish antibody specificity for the human platelet TXA2/PGH2 receptor protein. Functional analysis demonstrated that neither antibody interfered with ADP- or U46619-induced platelet aggregation of [3H]SQ29,548 binding to the solubilized receptor. These results suggest that the antibody epitopes are separate from the TXA2/PGH2 binding domain. In summary, two specific anti-peptide antibodies have been raised against the human platelet TXA2/PGH2 receptor. These antibodies should prove to be of value in the further investigation of the platelet TXA2/PGH2 receptor.