Microscopic view on the polarization-resolved S-SHG intensity of the vapor/liquid interface of pure water.

Second harmonic generation (SHG) is a nonlinear optical phenomenon where two photons at the frequency ω combine to form a single photon at the second-harmonic frequency 2ω. Since that second-order process is very weak in bulk isotropic media, optical SHG responses of interfaces provide a powerful and versatile technique to probe the molecular structure and dynamics of liquid interfaces. Both local dipole contributions and non-local quadrupole contributions can be interesting to investigate different properties of the interface, such as the molecular orientation or the charge density. However, a major difficulty is to comprehend the link between the S-SHG intensity and molecular details. This article reports a numerical approach to model the polarization-resolved SHG intensities of a model vapor/liquid interface of pure water. The influence of the interfacial local environment on the hyperpolarizability is taken into account using quantum mechanical/molecular mechanics calculations. The numerical predictions are in very good agreement with experiments. We detail the hypotheses made during the modeling steps and discuss the impact of various factors on the modeled SHG intensities, including the description of the exciting field in the interfacial layer, the effect of neighboring molecules on the second-harmonic polarization, and the presence of an additional static electric field at the interface.

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.

Abnormal platelet response to thromboxane A2.

To determine the pathogenetic mechanism of a hereditary primary platelet release disorder, arachidonic acid metabolism via the cyclooxygenase pathway was investigated. The propositus' platelets exhibited defective release reaction and second-wave aggregation when stimulated by sodium arachidonate or U46619, a thromboxane A2 (TXA2) agonist. The lack of platelet response to U46619 suggested that the defect was beyond the thromboxane synthetase level. Furthermore, thromboxane B2 (TXB2) formation in the propositus' platelets (558.52 ng/10(8) platelets) was within the normal range (574.29 +/- SD 27.39 ng/10(8) platelets) and TXA2 formation appeared to be adequate for aggregating normal platelets. The results were indicative of an abnormal platelet response to TXA2. Failure of the propositus' platelets to aggregate in response to TXA2 formed in normal platelet-rich plasma induced by arachidonate confirmed this notion. To gain further insight, platelet cyclic (c) AMP content was determined. Prostacyclin induced a significant elevation of the propositus' platelet cAMP level comparable to normal values. U46619 suppressed prostaglandin I2-induced cAMP elevation in normal subjects but had no such effect in the patient. We conclude that the primary release disorder observed in this kindred is due to an abnormal platelet respnse to TXA2 possibly because of TXA2/PGH2 receptor abnormalities.

Modulation of Ca2+ fluxes in isolated platelet vesicles: effects of cAMP-dependent protein kinase and protein kinase inhibitor on Ca2+ sequestration and release.

In the present study, we investigated the role of cAMP-dependent protein kinase in the process of Ca2+ uptake and release from platelet-derived membrane vesicles enriched in the dense tubular system. It was found that these membrane vesicles contain endogenous cAMP-dependent protein kinase and that stimulation of protein kinase by cAMP resulted in the phosphorylation of a single protein band (22 kDa). Addition of cAMP-dependent protein kinase produced effects on vesicle Ca2+ accumulation which were dependent on the Ca2+ concentration in the incubation medium. Specifically, at low extravesicular Ca2+ concentrations, cAMP-dependent protein kinase (10-100 micrograms/ml) produced a dose-dependent stimulation of Ca2+ uptake, however, a similar stimulation was not observed at high extravesicular Ca2+ concentrations. When endogenous protein kinase was blocked by the addition of protein kinase inhibitor, (2-160 nM) there was a dose-dependent inhibition of Ca2+ uptake at both low and high concentrations of extravesicular Ca2+. Furthermore, the addition of protein kinase inhibitor at steady state caused a rapid and dose-dependent release of vesicle-accumulated Ca2+. Studies on the phosphorylation profile of vesicle protein indicated that protein kinase inhibitor (80 and 160 nM) was capable of inhibiting the phosphorylation of the 22-kDa protein within 15 s. Finally, the ability of thromboxane A2 to cause Ca2+ release was inhibited by the addition of cAMP-dependent protein kinase (1 mg/ml). These findings suggest that cAMP-dependent protein kinase is not only a major determinant in the accumulation of Ca2+ by the dense tubular system, but may play an important role in the process of intraplatelet Ca2+ release by physiologic agents such as thromboxane A2.

The thromboxane antagonist, 13-azaprostanoic acid, inhibits arachidonic acid-induced Ca2+ release from isolated platelet membrane vesicles.

In the present study we investigated the ability of the arachidonic acid metabolites, prostaglandin H2 and thromboxane A2, to release Ca2+ from isolated platelet vesicles. The vesicles were prepared through modification of previously described procedures. 45Ca uptake and release were determined by Millipore filtration and isotope counting of the filter paper. Incubation of the vesicles (25 degrees C) with 50 microM CaCl2 (plus 45Ca) resulted in the accumulation of 13 nmol Ca2+ per mg of protein under steady-state conditions. Addition of arachidonic acid (25 microM) resulted in a 42% release of the accumulated Ca2+ and the production of 150 ng thromboxane B2/mg protein. Pretreatment of the vesicles with indomethacin (4 microM) completely inhibited arachidonic acid-induced Ca2+ release and reduced thromboxane B2 synthesis by 82%. Pretreatment of the vesicles with the specific thromboxane A2/prostaglandin H2 antagonist, 13-azaprostanoic acid (20 microM), also resulted in complete inhibition of Ca2+ release but no inhibition of thromboxane B2 production. Addition of prostaglandin H2 (0.3 microM) to the platelet vesicles produced a significant release of Ca2+ only in the presence of the adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (100 microM). This Ca2+ release was totally blocked by 13-azaprostanoic acid (20 microM). The thromboxane synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I, 3.6 microM), in the presence of 2',5'-dideoxyadenosine, only slightly inhibited Ca2+ release in response to added prostaglandin H2, even though thromboxane B2 production was blocked by 95%.

Movement of sodium into human platelets.

Addition of ADP induces platelets in plasma to undergo shape change from a disc to a spiny sphere and to develope adhesiveness, i.e. to aggregate. The aggregation of human platelets by ADP is associated with a net uptake of Na+. The present experiments demonstrate that the induction of shape change by ADP in acidified or EGTA-treated plasma conditions which inhibit aggregation, is also associated with a movement of Na+ into platelets. When ADP-induced platelet shape change and aggregation is inhibited by prostaglandin E1 Na+ uptake is also blocked. Platelets aggregated by epinephrine do not take up Na+. In a manner analogous to the effect of ADP, polylysine also induces Na+ uptake during aggregation. Vasopressin, in a manner analogous to epinephrine, induces aggregation without Na+ uptake. The increase in platelet Na+ resulting from ouabain inhibition of Na+ efflux induces an increase in the aggregation response to ADP and to epinephrine.

Movement of sodium into human platelets induced by ADP.

1. In normal human platelets the concentrations of Na+ and K+ were 42.1 +/- 4.3 and 98.8 +/- 3.7 mequiv/l of platelet water respectively (mean +/- S.E. of 22 samples). 2. When platelet-rich plasma was incubated with 22Na+ at 37 degrees C for 2-3 h an increase in platelet Na+ concentration was found which was significant after 210 min. Platelet K+ concentration did not change significantly. The platelet 22Na+ radioactivity increased faster than did the total Na+ suggesting a Na+o-Na+ exchange process in unactivated platelets. 3. Addition of ADP to platelet-rich plasma resulted in platelet aggregation and a rapid rise (within seconds) in 22Na+-radioactivity within the platelets and after 300 s this increase diminished toward control levels. 4. Under the same experimental conditions, ADP did not bring about an increase of 36Cl- in the platelets. 5. Ouabain (10-(6) M) added to platelet-rich plasma induced an increase in Na+ concentration and 22Na+ radioactivity in the platelets, as well as a decrease in K+ concentration. ADP produced a further increase in 22Na+, which did not return toward control values, in the presence of ouabain. 6. The association of an increase in 22Na+ but not of 36Cl- accompanying aggregation by ADP suggests a selective mechanism for the movement of Na+ into platelets rather than a movement of NaCl together with water under an osmotic gradient.

Specific binding of the thromboxane A2 antagonist 13-azaprostanoic acid to human platelet membranes.

In the present study we characterized the interaction between the thromboxane A2/prostaglandin H2 antagonist, trans-13-azaprostanoic acid (13-APA), and isolated human platelet membranes. In these studies, we developed a binding assay using trans [3H] 13-APA as the ligand. It was found that trans [3H] 13-APA specific binding was rapid, reversible, saturable and temperature dependent. Scatchard analysis of the binding data yielded a curvilinear plot which indicated the existence of two classes of binding sites: a high-affinity binding site with an estimated dissociation constant (Kd) of 100 nM; and a low-affinity binding site with an estimated Kd of 3.5 microM. At saturation, approximately 1 pmol/mg protein of [3H] 13-APA was bound to the high affinity site. In order to further characterize the nature of the [3H] 13-APA binding site, we evaluated competitive binding by cis 13-APA, cis 15-APA, prostaglandin F2 alpha, U46619, 6-ketoprostaglandin F1 alpha and thromboxane B2. It was found that the [3H] 13-APA binding site was stereospecific and structurally specific. Thus, the cis isomer of 13-APA exhibited substantially reduced affinity for binding. Furthermore, the prostaglandin derivatives, thromboxane B2 and 6-ketoprostaglandin F1 alpha, which do not possess biological activity, also did not compete for [3H] 13-APA binding. On the other hand, U46619 which acts as a thromboxane A2/prostaglandin H2 mimetic, and prostaglandin F2 alpha which acts as a thromboxane A2/prostaglandin H2 antagonist, both effectively competed for [3H] 13-APA binding. These findings indicate that trans 13-APA binds to a specific site on the platelet membrane which presumably represents the thromboxane A2/prostaglandin H2 receptor.

Increased membrane-bound calcium in platelets of hypertensive patients.

The fluorescent indicator chlortetracycline was used to estimate membrane-bound calcium in mild, untreated hypertensive patients (n = 39) and normotensive controls (n = 42). All participants were black. After incubation with chlortetracycline, platelet-rich plasma was centrifuged into a pellet and fluorescence was measured with a microspectrofluorometer. At an interval of 45 minutes mean fluorescence values were 11% higher in the hypertensive than in the normotensive group (567 +/- 95 vs. 512 +/- 100 counts/sec, p less than 0.02). With both groups of participants combined, a correlation of borderline statistical significance was noted between diastolic blood pressure and chlortetracycline fluorescence (r = 0.213, p = 0.056). In parallel experiments, sodium and potassium concentrations were measured in red blood cells. Intracellular sodium was also significantly higher in the hypertensive group (p less than 0.01). These data indicate that the total cell burden of calcium is increased in the platelets of hypertensive individuals, possibly a result of abnormal cell metabolism of calcium, and further suggest that circulating platelets in hypertensive individuals may be in a hyperaggregable state.

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.

Eicosapentaenoic acid and docosahexaenoic acid are antagonists at the thromboxane A2/prostaglandin H2 receptor in human platelets.

The present study investigated the mechanism by which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibit platelet activation induced by thromboxane A2. DHA was found to be more potent than EPA in blocking platelet aggregation induced by the stable thromboxane A2 mimetic, U46619. Furthermore, this inhibition by DHA or EPA was competitive. Binding studies using 3H-U46619 demonstrated that both EPA and DHA interact with the platelet thromboxane receptor. The potency of the inhibition of binding corresponded with that seen for the inhibition of aggregation. These results suggest that thromboxane receptor antagonism may be an important mechanism by which EPA and DHA modulate platelet reactivity in vivo.

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets.

A photoactive iodoarylazide derivative (I-APA-PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor-binding studies using [3H]U46619, I-APA-PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I-APA-PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding ratio-iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2-binding protein in human platelets.

Azaprostanoic acid derivatives. Inhibitors of arachidonic acid induced platelet aggregation.

A series of 13-azaprostanoic acids (4a-h) and a 15-azaprostanoic acid (11a) have been prepared. Synthesis of the 15-aza derivative is based on a novel transformation of a ketone to an N-substituted ethylenamine using a formylmethylimino phosphate derivative. Several of the azaprostanoic acid derivatives were found to be potent inhibitors of platelet aggregation induced by arachidonic acid, whereas no effect was observed on ADP-induced primary aggregation, indicating blockade of the platelet arachidonic acid cascade. The compounds do not inhibit bovine cyclooxygenase activity and are postulated as acting beyond the synthesis of the prostaglandin endoperoxides. The inhibitory effect of the 13-aza series is highly sensitive to both stereochemistry and length of the amino side chain. Any deviation from the natural prostaglandin skeletal arrangement results in decreased biological activity.

PgH2 analogs as potential antiplatelet derivatives.

Previous observations implicating PgH2 as a direct activator of platelets suggested that derivatives of U46619, a well-characterized TxA2 receptor agonist having structural homology with PgH2, might possess antiplatelet activity. The present work describes the synthesis of [1S-(1 alpha,2 beta,3 alpha,4 alpha)]-3-[(tetrahydropyranyloxy)methyl]- 2-[2-[(triphenylmethyl)oxy]ethyl]-5-oxabicyclo[2.2.1]heptane (14) a potentially useful intermediate for the synthesis of various epoxymethano derivatives. The latter was converted to [1S-(1 alpha,2 beta (Z),3 alpha,4 alpha)]-7-[3-[[2- [(phenylamino)carbonyl]-hydrazino]methyl]-5-oxabicylo[2.2.1]hept-2 - yl]-5-heptenoic acid (23), an epoxymethano derivative of PgH2 containing a hydrazide lower side chain as previously used in the TxA2 antagonist, SQ 29,548. The intermediate 14 was also converted to [1S-(1 alpha,2 beta (Z),3 alpha,4 alpha)]-7- [3-[(hexylamino)methyl]-5-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (25) which contained a simple aza side chain as used in earlier antagonists. Derivatives 23 and 25 appeared to be specific antagonists of the human platelet TxA2 receptor as evidenced by their inhibition of U46619 (1.5 microM) induced aggregation of human platelet rich plasma (IC50 = 22 and 7 microM, respectively), while having little effect on ADP (2 microM) induced aggregation at much higher concentrations. In addition, one of these derivatives, the bicycloamine 25, was shown to compete for [3H]U46619 binding to washed human platelets with an IC50 value of 25 microM, supporting the notion that these derivatives were acting at the thromboxane receptor. However, the potency of these derivatives was less than for previously reported TxA2 antagonists, suggesting that simple linear combinations of functionality from molecules active at the human platelet thromboxane receptor will be of limited predictive value.

2-(6-carboxyhexyl)cyclopentanone hexylhydrazone. A potent and time-dependent inhibitor of platelet aggregation.

Two new azaprostanoids, a hydrazone (3) and hydrazide (4), have been prepared by the condensation of 2-(6-carboxyhexyl)cyclopentanone with n-hexylhydrazine and caproic acid hydrazide. Preliminary results with the stable hydrazide 4 indicate that it inhibits arachidonic acid (AA) induced human platelet aggregation and that, unlike 13-azaprostanoic acid (1), its site of action is at the cyclooxygenase level. Results with the unstable hydrazone derivative 3 indicate it to be a potent and time-dependent inhibitor of AA-induced human platelet aggregation, with its site of action also at the cyclooxygenase level.

Preparation and biological evaluation of a potential photoaffinity label for the prostaglandin H2/thromboxane A2 receptor.

Two aromatic azides (24 and 26) were prepared as potential photoaffinity probes for the PGH2/TXA2 receptor. The compounds are based on the well-characterized PGH2/TXA2 receptor antagonist 13-azaprostanoic acid, with the terminus of its lower side chain replaced with phenoxy (24) or benzyl (26) azide functionality. The two compounds were shown to irreversibly inhibit platelet function after photolysis and resuspension. However, of the two aromatic azides, only the benzyl derivative 26 appeared to be selective for the prostaglandin pathway. The latter compound was also prepared as the aromatic 125I (29) derivative, which may ultimately prove useful as a labeled probe for the identification and isolation of the putative TXA2/PGH2 receptor.

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.

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.

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.