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Biomedical subjects

J A May

Publications and source records attributed to J A May.

At least 19 recordsLinked to original sources
PubMed2004

Photoelectron spectroscopic and electronic structure studies of CH(2)O bonding and reactivity on ZnO surfaces: steps in the methanol synthesis reaction.

Adsorption of CH(2)O on ZnO(0001) has been investigated using XPS, NEXAFS, variable-energy photoelectron spectroscopy (PES), and density functional theory (DFT) calculations. CH(2)O is chemisorbed on the (0001) surface at 130 K. Its C1s XPS peak position at 292.7 eV and NEXAFS sigma shape resonance at 302.6 eV are consistent with an eta(1) bound surface geometry. Geometry optimized DFT calculations also indicate that CH(2)O is bound to the Zn(II) site in an eta(1) configuration through its oxygen atom. The variable-energy PES of the eta(1) bound CH(2)O/ZnO(0001) complex exhibits four valence band features at 21.2, 16.4, 13.8, and 10.7 eV below the vacuum level providing an experimental and theoretical description of this surface interaction. Annealing the ZnO(0001)/CH(2)O surface complex to 220 K decomposes the chemisorbed CH(2)O, producing formyl (291.5 eV), methoxide (290.2 eV), and formate (293.6 eV) intermediates. Thus this reaction coordinate involves the conversion of an oxygen bound formaldehyde to a carbon bound formyl species on ZnO(0001). Only formate is formed on the ZnO(100) surface. DFT is used to explore surface intermediates and the transition state in the methanol synthesis reaction (MSR). The bonding interactions of H(2), CO, CH(3)O(-), HCO(-), and trans-HCOH to the ZnO(0001) surface are elucidated using geometry optimization. H(2) was found to be heterolytically cleaved on the ZnO(0001) surface, and carbon monoxide, formyl, and methoxide are calculated to be eta(1) bound. These results are consistent with observed metal oxide surface reactivity where heterolytic bond cleavage is dominant. The oxygen atom in the bound formyl was found to be activated for attack by a proton. This results in the planar eta(1) bound trans-HCOH surface species. The transition state in the gas phase rearrangement of trans-HCOH to formaldehyde was calculated to have a barrier of 31 kcal/mol. The correlation diagram for this rearrangement in the gas phase indicates that configuration interaction at the crossing of two levels helps to lower the barrier. A transition state calculation was also performed for this rearrangement on the ZnO(0001) surface. The surface transition state geometry is significantly different than the gas phase. The surface geometry is no longer planar (23 degrees dihedral angle) and is displaced parallel to the surface. Interaction with the Zn(II) site at the crossing of surface bound CH(2)O and trans-HCOH levels further lowers the barrier to rearrangement relative to gas phase by 9 kcal/mol. The rearrangement of trans-HCOH (carbon bound) to CH(2)O (oxygen bound) on ZnO(0001) was calculated to be the overall barrier of the MSR reaction.

Journal Article↗
PubMed2004

Quantitation of platelet aggregation and microaggregate formation in whole blood by flow cytometry.

Platelet aggregation and microaggregate formation were measured in samples of stirred whole blood by flow cytometry. Blood samples were stirred in a multi-sample agitator with ADP, fixed and labelled with a platelet-specific CD42a-FITC fluorescent antibody. The blood was then diluted and applied directly to a flow cytometer. Platelets were identified using a gating procedure based on their expression of CD42a and then quantified. Aggregation was monitored as a fall in the number of single platelets. Both reversible and irreversible aggregation responses to ADP were determined and these were found to correlate directly with aggregation responses determined using a well-established single platelet counting technique using the Ultra-Flo 100 Whole Blood Platelet Counter. We found from flow cytometry that ADP-induced aggregation was coupled with a transient formation of platelet microaggregates over the initial 60 s following ADP addition. Assessment of single platelet loss by flow cytometry was found to be a reliable way of monitoring aggregation responses and provided new information on rapid microaggregate formation in ADP-stimulated blood.

Adenosine Diphosphate↗
PubMed2002

Interactions of platelets with Synthocytes, a novel platelet substitute.

A platelet substitute, Synthocytes, is being developed for the prevention and treatment of thrombocytopenia. Synthocytes are composed of fibrinogen adsorbed on heat stabilised albumin microcapsules of defined size. The purpose of this study was to perform experiments in vitro to investigate the capacity of Synthocytes to interact with platelets, one of the means through which Synthocytes may contribute to haemostatic plug formation in vivo. Synthocytes were found to interact with platelets as shown by platelet aggregation assays and measurements of [(14)C]5HT release from platelets in whole blood and platelet-rich plasma. Platelet-Synthocytes co-aggregate formation was demonstrated directly using flow cytometry and the presence of activated platelets in these co-aggregates was demonstrated using an antibody to P-selectin. Synthocytes enhanced platelet responsiveness to conventional aggregating agents such as ADP. Indeed, antagonists of the action of ADP on platelets inhibited the direct effects of Synthocytes on platelets in whole blood, as did a GPIIb/IIIa antagonist. Enhancement of annexin V binding was also observed, indicative of increased pro-coagulant activity. Experiments performed with control microcapsules (lacking fibrinogen) confirmed the importance of fibrinogen in the interactions that occurred. The results suggest that fibrinogen on the surface of Synthocytes can interact with GPIIb/IIIa on platelets to induce platelet activation, secretory activity and aggregation, and that ADP contributes to this process. This initial interaction renders platelets more susceptible to the stimulatory effects of other platelet-activating agents. It is considered likely that in the clinical setting of thrombocytopenia any interaction between Synthocytes and residual platelets that are present may contribute to primary haemostasis.

Adenosine Diphosphate↗
PubMed2000

2H-Thieno[3,2-e]- and [2,3-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides as ocular hypotensive agents: synthesis, carbonic anhydrase inhibition and evaluation in the rabbit.

Novel non-chiral 2H-thieno[3,2-e]- and [2,3-e]-1,2-thiazine-6-sulfonamide 1,1-dioxides were synthesized for evaluation as potential candidates for the treatment of glaucoma. All of the compounds prepared were potent high affinity inhibitors of human carbonic anhydrase II, Ki < 0.5 nM. Additionally, inhibition of recombinant human carbonic anhydrase IV was determined for selected compounds; these were shown to be moderate to potent inhibitors of this isozyme with IC50 values ranging from 4.25 to 73.6 nM. Of the compounds evaluated for their ability to lower intraocular pressure in naturally hypertensive Dutch-belted rabbits, 5a, 17a3, 17b1, 17b2, 17h2 and 17i1 showed significant efficacy (> 20% decrease) in this model following topical ocular administration.

Animals↗
PubMed2000

The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity.

Adenosine diphosphate (ADP) is an important platelet agonist and ADP released from platelet dense granules amplifies responses to other agonists. There are three known subtypes of ADP receptor on platelets: P2X(1), P2Y(1) and P(2T) receptors. Sustained ADP-induced aggregation requires co-activation of P2Y(1) and P(2T) receptors. AR-C69931MX, a selective P(2T) receptor antagonist and novel antithrombotic agent, was studied to characterize further the function of the P(2T) receptor. The roles of the P2Y(1) receptor and thromboxane A(2) were assessed using the selective P2Y(1) antagonist A2P5P and aspirin respectively. Aggregation was measured by whole blood single-platelet counting and platelet-rich plasma turbidimetry, using hirudin anticoagulation. Dense granule release was estimated using ([14)C]-5-hydroxytryptamine (HT)-labelled platelets. Ca(2+) mobilization, P-selectin expression, Annexin V binding and microparticle formation were determined by flow cytometry. P(2T) receptor activation amplified ADP-induced aggregation initiated by the P2Y(1) receptor, as well as amplifying aggregation, secretion and procoagulant responses induced by other agonists, including U46619, thrombin receptor-activating peptide (TRAP) and collagen, independent of thromboxane A(2) synthesis, which played a more peripheral role. P(2T) receptor activation sustained elevated cytosolic Ca(2+) induced by other pathways. These studies indicate that the P(2T) receptor plays a central role in amplifying platelet responses and demonstrate the clinical potential of P(2T) receptor antagonists.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2000

Effects of cytochalasin H, a potent inhibitor of cytoskeletal reorganisation, on platelet function.

Platelets contain a well-developed and dynamic cytoskeleton composed mainly of actin and actin-associated proteins. Upon platelet activation there is rapid polymerisation of actin and a marked reorganisation of the platelet cytoskeleton. Cytochalasins are agents that interfere with the polymerisation of actin, and it has recently been discovered that cytochalasin H (CyH) is particularly effective as an inhibitor of the cytoskeletal reorganisation that occurs in platelets following activation by adenosine diphosphate (ADP). Here we have used CyH to inhibit platelet cytoskeletal reorganisation and to determine its effects on various aspects of platelet function. Experiments were performed in hirudinized platelet-rich plasma (PRP) or whole blood obtained from human volunteers. PRP was treated with 10 microM CyH or vehicle, then activated by ADP. The effect of CyH on cytoskeletal reorganisation was determined by SDS-PAGE of the Triton X-100 insoluble cytoskeletons and quantitated by densitometry. Platelet aggregation and aggregate stability in PRP were measured by monitoring changes in light absorbance; aggregation was measured in whole blood via platelet counting. Shape change, P-selectin expression and changes in intracellular calcium were measured using flow cytometry. CyH prevented the normal incorporation of actin, alpha-actinin and actin-binding protein into the cytoskeleton that occurred following ADP activation, and incorporation of myosin was markedly reduced. Aggregation was only partially inhibited but, more dramatically, the rate of disaggregation following addition of certain agents that interfere with fibrinogen binding to glycoprotein IIb/IIIa on the surface of platelets was markedly increased. The ADP-induced shape change was also inhibited. CyH had no effect on calcium mobilisation. Curiously, expression of P-selectin was potentiated by CyH, suggesting a modulatory role of the cytoskeleton in platelet secretory activity. The results suggest that cytoskeletal reorganisation plays an important role in platelet shape change and aggregation and contributes in a major way to the stability of the aggregates that form.

Adenosine Diphosphate↗
PubMed1999

A mouse fibroblast line cycles between monolayer and spheroid forms, regulates Met and HGF expression, and releases an attachment and growth-promoting substance.

A subline of mesoderm-derived mouse NIH3T3 fibroblasts was selected for its ability to proliferate in serum-free media. This cell line (SFDH) grows as a monolayer at low density and spontaneously forms dense, multicellular spheroids at high density. Spheroid formation can also be induced by the addition of dexamethasone, polybrene, or heparin. Spheroids eventually detach from the substrate, but will reattach and re-form monolayers when transferred to fresh culture vessels and media, repeating the cycle again upon reaching high density. Thin section analysis of spheroids shows morphologically-distinct regions of cells, including an attenuated outer surface and a cuboidal interior with occasional lumen-like areas. Over time in culture, spheroids express increasing levels of met, the Met ligand-SF/HGF and cytokeratin, an epithelial marker, in comparison to monolayers. Both monolayer and spheroid-derived cells are rapidly tumorigenic in nude mice. Media conditioned by SFDH cells contain factors that stimulate growth and attachment of a variety of tumorigenic and non-tumorigenic cell lines, inducing cells to divide in serum-free media for up to 14 days when plated on tissue culture-treated and nontreated plastic surfaces pre-coated with SFDH conditional media. The growth-stimulating activity fractionates as a single peak over a sepharose column in the presence of 6 m urea, and sediments as a high molecular weight complex. Growth-stimulating activity can be neutralized by several antisera specific for hepatocyte growth factor, and the same sera recognize a novel approximately 37 kD protein in active supernatants. The cyclic, continuous nature of alternating monolayer and spheroid forms makes this cell line appropriate for studying changing gene expression patterns in progressive cell-cell/cell-matrix interactions.

3T3 Cells↗
PubMed1999

Comparison of antiplatelet activity of microencapsulated aspirin 162.5 Mg (Caspac XL), with enteric coated aspirin 75 mg and 150 mg in patients with atherosclerosis.

AIMS: A new formulation, low dose microencapsulated aspirin, permits slow absorption of aspirin and presystemic acetylation of platelet cyclo-oxygenase within the portal circulation, potentially avoiding deleterious effects on gastric and systemic prostaglandin synthesis. The objective of this study was to determine whether the administration of microencapsulated aspirin was as effective as enteric coated (EC) aspirin as an inhibitor of platelet function in patients with atherosclerosis. METHODS: One hundred and four patients were enrolled and randomised after a run in period of at least 14 days on aspirin EC 75 mg (day 0), to receive either microencapsulated aspirin 162.5 mg (n=34), aspirin EC 150 mg (n=36) or continue on aspirin EC 75 mg (n=34) for 28 days. Serum thromboxane B2 and collagen-induced platelet aggregation and release of 5-hydroxytryptamine (EC50 values) were measured on days 0 and 28. Aggregation/release EC50s were then repeated in the presence of a large dose of aspirin added in vitro to determine the EC50 at the maximum level of platelet inhibition. RESULTS: Median thromboxane B2 levels were low after 14 days run-in therapy with aspirin EC 75 mg, but significant further reductions were seen on day 28 in patients randomised to microencapsulated aspirin 162.5 mg (P=0.0368) and aspirin EC 150 mg (P=0.0004) compared with those remaining on aspirin EC 75 mg. Median EC50 s on day 28 showed small but significant increases from baseline (day 0) in aggregation in patients randomised to microencapsulated aspirin 162.5 mg (0.62-0.85, P=0.0482) and in both aggregation and release in patients randomised to aspirin EC 150 mg (0.95-1.20, P=0.0002, 8.4-11.7, P<0. 0001, respectively) signifying enhanced antiplatelet activity. No changes were seen in patients continuing on aspirin EC 75 mg. Results following addition of high dose aspirin in vitro suggest that mechanisms other than thromboxane synthesis may be operative in the long term effects of microencapsulated aspirin 162.5 mg and aspirin EC 150 mg over aspirin EC 75 mg. CONCLUSIONS: The results show good inhibition of thromboxane B2 synthesis and subsequent platelet activity by all preparations of aspirin, although both microencapsulated aspirin 162.5 mg and aspirin EC 150 mg are slightly more effective than aspirin EC 75 mg. A randomised trial is now required to determine whether microencapsulated aspirin is associated with fewer gastric side-effects.

Arteriosclerosis↗
PubMed1999

A whole blood assay of inhibition of platelet aggregation by glycoprotein IIb/IIIa antagonists: comparison with other aggregation methodologies.

We have used a whole blood single-platelet counting assay (WB-SPC) that is sensitive to microaggregation for monitoring GPIIb/IIIa antagonists and have compared this with other methodologies. In vitro effects of the GPIIb/IIIa antagonist fradafiban on ADP-induced platelet aggregation were determined using WBSPC and PRP turbidimetry, comparing citrate and hirudin anticoagulation. Fradafiban was a more potent inhibitor of aggregation assessed by PRP turbidimetry compared to WBSPC. Citrate showed only a trend towards enhancing fradafiban potency (p = 0.087). Citrated blood from 8 patients with unstable angina, randomised to receive oral lefradafiban (the oral prodrug of fradafiban) or placebo, was studied before and during treatment using WBSPC, PRP turbidimetry, impedance aggregometry and Rapid Platelet Function Assay (RPFA, Accumetrics). RPFA, PRP turbidimetry and WBSPC measurements correlated well. Impedance aggregometry responses were oversensitive to GPIIb/IIIa blockade. WBSPC was most discriminating at high levels of inhibition and offered a rapid means of monitoring GPIIb/IIIa antagonist effect within the therapeutic range of inhibition.

Adolescent↗
PubMed1998

Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: molecular basis of isozyme-drug discrimination.

Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-angstroms resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some local structural differences are found in the active site resulting from amino acid sequence differences in the "130's segment" and the residue-63 loop (these may affect the nearby catalytic proton shuttle, His-64). Similar to human CAIV, the C-terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.

Amino Acid Sequence↗
PubMed1998

Structural analysis of inhibitor binding to human carbonic anhydrase II.

X-ray crystal structures of carbonic anhydrase II (CAII) complexed with sulfonamide inhibitors illuminate the structural determinants of high affinity binding in the nanomolar regime. The primary binding interaction is the coordination of a primary sulfonamide group to the active site zinc ion. Secondary interactions fine-tune tight binding in regions of the active site cavity >5 A away from zinc, and this work highlights three such features: (1) advantageous conformational restraints of a bicyclic thienothiazene-6-sulfonamide-1,1-dioxide inhibitor skeleton in comparison with a monocyclic 2,5-thiophenedisulfonamide skeleton; (2) optimal substituents attached to a secondary sulfonamide group targeted to interact with hydrophobic patches defined by Phe131, Leu198, and Pro202; and (3) optimal stereochemistry and configuration at the C-4 position of bicyclic thienothiazene-6-sulfonamides; the C-4 substituent can interact with His64, the catalytic proton shuttle. Structure-activity relationships rationalize affinity trends observed during the development of brinzolamide (Azopt), the newest carbonic anhydrase inhibitor approved for the treatment of glaucoma.

Binding Sites↗
PubMed1998

GPIIb-IIIa antagonists cause rapid disaggregation of platelets pre-treated with cytochalasin D. Evidence that the stability of platelet aggregates depends on normal cytoskeletal assembly.

Platelet activation is accompanied by changes in the composition of the platelet cytoskeleton with rapid incorporation and displacement of certain proteins. Here we have inhibited cytoskeletal assembly by pretreating platelets with cytochalasin D (CyD) and investigated the effect on the stability of the aggregates that form. The experiments were performed in both citrated and hirudinized platelet-rich plasma (PRP) and aggregation was induced by adenosine diphosphate (ADP), collagen, the TXA2-mimetic U46619 and adrenaline. Platelets in the aggregates that formed, underwent rapid disaggregation on addition of EDTA or a GpIIb-IIIa antagonist such as MK-852 and GR144053F, all of which are agents that interfere with the ability of fibrinogen to interact with GpIIb-IIIa. This was the case irrespective of the aggregating agent used and occurred in both citrated and hirudinized PRP. In contrast, the rate of disaggregation brought about by some other agents, iloprost and ARL 66096, appeared to be unaffected by CyD. Information was also obtained on the effects of CyD on the cytoskeletal changes brought about by ADP and the effects on the cytoskeleton of subsequent addition of M K-852. The results show that CyD retards the incorporation of certain proteins (actin, myosin, alpha -actinin, actin binding protein and a 66 K protein) into the cytoskeleton and that subsequent addition of MK-852 results in rapid displacement of some of these with re-incorporation of a 31 K protein. The results suggest that the early changes in the cytoskeleton following platelet activation contribute to the stability of the aggregates that form, and that interference with these early changes results in aggregates that are easily disassembled by agents that interfere with GpIIb-IIIa-fibrinogen complex formation.

Journal Article↗
PubMed1997

Platelet responses to several agonists and combinations of agonists in whole blood: a placebo controlled comparison of the effects of a once daily dose of plain aspirin 300 mg, plain aspirin 75 mg and enteric coated aspirin 300 mg, in man.

Platelet responses to several agonists and combinations of agonists have been measured in whole blood from healthy volunteers. We have determined the effects of once daily treatment for five days with plain aspirin 300 mg, plain aspirin 75 mg, enteric coated aspirin 300 mg or placebo. Measurements were made of platelet aggregation (using a platelet counting technique) and the release reaction (14C-5HT release from pre-labelled platelets). The extents of these responses before aspirin administration depended on the agonist used. ADP, adrenaline and PAF failed to induce any 14C-5HT release in most subjects, but combinations of these agonists acted synergistically to produce extensive 14C-5HT release. All three aspirin preparations reduced the extent of the platelet responses to most agonists: platelet aggregation induced by collagen, ristocetin and arachidonate and 14C-5HT release induced by collagen, streptokinase, and various combinations of ADP, adrenaline and PAF. None of the preparations had any effect on the aggregation that occurred in the absence of an agonist (spontaneous aggregation), but they all reduced streptokinase-induced aggregation to control (spontaneous) levels, and abolished the 14C-5HT release induced by arachidonate and by ristocetin. All three aspirin preparations were equally effective after two daily doses. No further inhibition of platelet responses was obtained after five daily doses. Plain aspirin 300 mg achieved its maximal effect after only a single dose, but enteric coated aspirin 300 mg (and sometimes plain aspirin 75 mg) produced sub-maximal inhibition after only a single dose. Parallel investigations on the effects of these aspirin regimes on gastric mucosal prostaglandin E2 synthesis and gastroduodenal mucosal injury were performed. These results will be reported separately.

Adenosine Diphosphate↗
PubMed1996

Changes in the composition of the platelet cytoskeleton in response to ADP: effects of MK-852 and ARL 66096.

Platelet activation by adenosine diphosphate (ADP) results in an alteration in the composition of the cytoskeleton. Here we have determined the effects of MK-852 and ARL 66096 on the cytoskeletal changes that occur. MK-852 is a GPIIb/IIIa antagonist that inhibits aggregation by interfering with fibrinogen binding ARL 66096 is a P2T antagonist that selectively inhibits ADP-induced aggregation. Neither agent inhibits the shape change response. Experiments were performed in hirudinized platelet-rich plasma. Platelet activation led to a significant and sustained increase in the cytoskeletal content of actin binding protein (ABP), myosin, alpha-actinin, a 66K protein and actin, and a significant decrease in a 31K protein. In the presence of MK-852 there was no increase in ABP or the 66K protein and no decrease in the 31K protein. The increase in myosin and alpha-actinin became reversible but there was still incorporation of actin into the cytoskeleton. In the presence of ARL 66096 there was no increase in ABP or the 66K protein and no decrease in the 31K protein. ARL 66096 also prevented incorporation of alpha-actinin and actin. As with MK-852, myosin incorporation became reversible. The results suggest that (1) myosin is incorporated into the cytoskeleton transiently during shape change, (2) ADP interaction with the ADP aggregation receptor (but not that for shape change) is associated with alpha-actinin and actin incorporation into the cytoskeleton, and (3) further changes that occur are consequent to fibrinogen binding and platelet aggregation.

Actinin↗
PubMed1995

Factors that contribute to spontaneous platelet aggregation and streptokinase-induced aggregation in whole blood.

When whole blood is stirred there is a "spontaneous" platelet aggregation (SPA) which is presumed to be caused by proaggregatory factors released from platelets and other blood cells. Adding streptokinase (SK) to stirred whole blood frequently increases the rate and extent of the platelet aggregation that occurs; this is likely to be via immune complex formation between SK and natural anti-SK antibodies leading to increased release of pro-aggregatory factors. In this investigation we have examined the effects of several inhibitors and antagonists in an attempt to identify the proaggregatory factors that contribute to both SPA and SK-induced aggregation (SKA) and to evaluate different means of inhibiting both processes. The effects of the inhibitors/antagonists were determined in vitro after adding them to citrated whole blood obtained from healthy volunteers. Platelet aggregation was measured using a platelet counting technique. Inhibition of both SPA and SKA by apyrase and by FPL 66096 (a P2T receptor antagonist) demonstrated the involvement of ADP in both processes. Inhibition by chlorpromazine indicated that the most likely source of the ADP is red cells. The effects of sulotroban (a TXA2 antagonist) indicated involvement of TXA2 in SKA but not in SPA. The lack of effect of specific antagonists at S2, alpha 2 and PAF receptors suggested lack of involvement of serotonin, catecholamines and platelet-activating factor in either SPA or SKA. Both SPA and SKA were potently inhibited by low concentrations of iloprost (a PGI2 analogue), but a high concentration of SIN-1 (a NO donor) was much less effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗