Patterns of helminth infections in Rattus rattus and Mus musculus from two Mayan communities in Mexico.

The black rat Rattus rattus and the house mouse Mus musculus are two commensal rodent species that harbour and shed zoonotic pathogens, including helminths. The aim of this survey was to study the helminth community and the patterns of infections in R. rattus and M. musculus from two Mayan communities in Mexico. Gastrointestinal helminths were isolated from 322 M. musculus and 124 R. rattus, including Gongylonema neoplasticum, Hassalstrongylus aduncus, Hassalstrongylus musculi, Hydatigera taeniaeformis metacestode, Hymenolepis diminuta, Nippostrongylus brasiliensis, Oligacanthorhynchidae gen. sp., Syphacia muris, Syphacia obvelata, Rodentolepis microstoma and Trichuris muris. The overall richness of helminths was seven in R. rattus and six in M. musculus. The results of generalized linear models showed that juvenile rodents had lower probabilities of being infected with G. neoplasticum, H. taeniaeformis and H. musculi than adult rodents. A positive association between the prevalence of S. muris and rat abundance was found. The intensity of infection with S. muris was higher in the rainy season than in the dry season; the opposite result was found for H. musculi infection. Male R. rattus harboured more S. muris specimens. The intensity of infection with T. muris was inversely associated with mouse abundance. The presence of the zoonotic H. diminuta, as well as H. taeniaeformis and R. microstoma in rodent populations indicates that there is risk of transmission, and that their entire life cycle occurs in the study area.

Effects on platelet function of an EP3 receptor antagonist used alone and in combination with a P2Y12 antagonist both in-vitro and ex-vivo in human volunteers.

EP3 receptor antagonists may provide a new approach to the treatment of atherothrombotic disease by blocking the ability of prostaglandin E2 (PGE2) to promote platelet function acting via EP3 receptors. DG-041 is an EP3 antagonist in the early stage of clinical development. Here, we quantitated effects on platelet function of DG-041 in-vitro and ex-vivo after administration to man when given alone and concomitantly with clopidogrel or clopidogrel and aspirin. With its unique mechanism of action, it was anticipated that DG-041 would potentiate inhibition of platelet function when given in combination with clopidogrel without materially increasing bleeding time. Initially, in-vitro studies were performed to determine inhibitory effects of DG-041 (3 µM) used alone or in combination with the P2Y12 antagonist cangrelor (1 µM), both without and with aspirin (100 µM). Platelet aggregation and P-selectin expression were measured in whole blood (n = 10) following stimulation with the thromboxane A2 (TXA2) mimetic U46619 (0.3 or 1 µM) in combination with either the EP3 agonist sulprostone (0.1 µM), or PGE2 (1 µM). DG-041 alone partially inhibited platelet function in-vitro, as did cangrelor. Addition of both DG-041 and cangrelor in combination provided significantly greater inhibition. An ex-vivo study was then performed using the same experimental approaches. This clinical study was a prospective, randomised, blinded (for DG-041/matching placebo), blocked, crossover study designed to compare the effects of DG-041, clopidogrel, or the combination of DG-041 with either clopidogrel or clopidogrel and aspirin. Healthy volunteers (n = 42) were randomly assigned to receive no background treatment, clopidogrel (300 mg loading dose plus 75 mg daily) or clopidogrel and aspirin (75 mg daily) for 10 days alongside DG-041 (200 mg twice daily) or placebo for 5 days, crossed over to placebo or DG-041 for the next 5 days. Platelet effects and bleeding time were measured at baseline, days 5 and 10. DG-041 partially inhibited platelet function ex-vivo, as did clopidogrel, while administration of both DG-041 and clopidogrel provided significantly greater inhibition. Administration of DG-041 alone did not increase bleeding time, and did not significantly affect the increased bleeding time seen with clopidogrel or clopidogrel with aspirin. Using these experimental approaches, the antiplatelet effects of DG-041 and a P2Y12 antagonist used alone and in combination can be determined both in-vitro and ex-vivo. Results show inhibitory effects of DG-041 on platelet function acting via EP3 receptor blockade, confirmed to be additional to those brought about by P2Y12 blockade. In both in-vitro and ex-vivo studies, aspirin neither promoted nor negated the effects of the other drugs.

The occurrence of the larval cestode Cysticercus fasciolaris in rodent populations from the Cuxtal ecological reserve, Yucatan, Mexico.

Cysticercus fasciolaris is the larval stage of the cestode Taenia taeniaeformis, whose definitive hosts are mainly cats. This larval stage uses a wide variety of small rodents, and occasionally birds and humans, as intermediate hosts. In the Yucatan, there are no reports of the presence of this cestode in animal populations. The aim of this study was to evaluate the occurrence of C. fasciolaris in rodent populations from the Cuxtal ecological reserve, Yucatan, Mexico. Trapping of rodents was conducted from October 2009 to April 2010 in 40 households in Molas, in which Sherman traps were placed both inside and outside backyards. Rodents were dissected to inspect the liver for the presence of the worm. To determine risk factors associated with infection, univariate analysis was performed using sex, age, species, trapping site, and season as independent variables. Variables with a P value <  0.2 were analysed using a logistic regression model. In this study, 411 individuals of six rodent species were trapped; Mus musculus was the most abundant (78%), followed by Rattus rattus (13%) and the wild species Peromyscus yucatanicus, Ototylomys phyllotis, Heteromys gaumeri and Reithrodontomys gracilis (9%). Only 7.5% (n = 31) of M. musculus and R. rattus were infected with C. fasciolaris (demonstrated by the presence of liver cysts) with a prevalence of 9.0% and 3.5%, respectively. Both adults and male mice were 4.33 and 3.46 (OR values) times more likely to have C. fasciolaris than juveniles and females respectively. We can conclude that in the Cuxtal Reserve, Yucatan, Mexico, the prevalence of C. fasciolaris is higher in M. musculus, and that adult males had a higher probability of infection. Wild species, mainly P. yucatanicus, were not found to be infected with the cestode, but its presence in the backyards of households could result in a potential risk of acquiring this infection.

Measurement of platelet P-selectin for remote testing of platelet function during treatment with clopidogrel and/or aspirin.

There is great interest in assessing the efficacy of treatment with clopidogrel and aspirin in patients with cardiovascular disease using procedures that can be used in a remote setting. Here we have established methods to assess the effects of clopidogrel and aspirin on platelets based on measurements of platelet P-selectin. Platelets were stimulated in whole blood by adding the combination of adenosine diphosphate and the TXA(2) mimetic U46619 (ADP/U4, designed to assess P2Y(12) inhibition) or the combination of arachidonic acid and epinephrine (AA/Epi, designed to assess COX-1 inhibition). The stimulated samples were then fixed using a fixative solution that provides stability for at least 9 days, and sent to a central laboratory for analysis of P-selectin by flow cytometry. Measurements were performed in blood from healthy volunteers and patients with cardiovascular disease. The inhibitory effects of clopidogrel and aspirin were assessed ex vivo and the effects of the direct acting P2Y(12) antagonist cangrelor and aspirin were assessed in vitro. Measurements of platelet aggregation were also performed for comparison. In healthy volunteers clopidogrel ex vivo and cangrelor in vitro markedly inhibited P-selectin expression induced by ADP/U4. Aspirin did not inhibit and did not interfere with the effects of clopidogrel or cangrelor using this test. There was very little overlap of results obtained in the absence and presence of clopidogrel or cangrelor. In contrast, over half of 42 patients with cardiovascular disease did not respond well to clopidogrel treatment, although cangrelor was still effective. Aspirin markedly inhibited P-selectin expression induced by AA/Epi. Clopidogrel had much less effect and did not interfere with the effects of aspirin. There was no overlap of results obtained in the absence and presence of aspirin. Aspirin provided near-complete inhibition in 29 of 30 patients with cardiovascular disease. Aggregometry measurements agreed well with the P-selectin data obtained ex vivo following both clopidogrel and aspirin treatment. It is concluded that measurements of P-selectin performed on fixed blood samples following platelet stimulation in whole blood in a remote setting can be used effectively to monitor the effects of clopidogrel and aspirin.

Acyl derivatives of coenzyme A inhibit platelet function via antagonism at P2Y1 and P2Y12 receptors: a new finding that may influence the design of anti-thrombotic agents.

We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca2+ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca2+ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y(1) receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca2+ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y(1) antagonist) and AR-C69931 (a P2Y(12) antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y(1) but also with partial antagonism at P2Y(12) receptors. Antagonism at P2Y(12) receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X(1) receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.

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.

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.

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.

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.

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.

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.

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.

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.

The composition of the platelet cytoskeleton following activation by ADP: effects of various agents that modulate platelet function.

Platelet activation by adenosine diphosphate (ADP) results in changes in the composition of the large cytoskeletal fragments that can be isolated following solubilization of platelets with Triton X-100 and low speed centrifugation. Here we have used several different agents that modify platelet responses to investigate some of the factors that affect these cytoskeletal changes. All the experiments involved use of hirudinized platelet-rich plasma in which TXA, synthesis and release of dense body constituents does not occur following platelet activation with ADP. ADP alone caused a significant and sustained increase in the cytoskeletal content of actin binding protein (ABP), myosin, α-actinin, a 66K protein and actin, and a significant decrease in a 31K protein. In the presence of MK-852 or GR 144053 (GpIIbDIIa antagonists), in samples merely left unstirred and in Glanzmann's thrombasthesenia, ADP produced no increase in ABP or the 66K protein and no decrease in the 31K protein. The increase in myosin and α-actinin became reversible but there was still incorporation of actin into the cytoskeleton. In the presence of ARL 66096 (a P(2T) purinoceptor antagonist that inhibits aggregation but not shape change) there was no increase in ABP or the 66K protein and no decrease in the 31K protein. ARL 66096 also prevented incorporation of α-actinin and actin. As with MK-852, myosin incorporation became reversible. Iloprost inhibited all the cytoskeletal changes, the effects of MgCI(2) were similar to those of MK-852, and acetylsalicylic acid (ASA) had no effect. In some experiments MK-852, ARL 66096, iloprost or MgCI, were added 0.5 min after the ADP. They all produced disaggregation and this was accompanied by reversal of the changes in the composition of the cytoskeleton that had occurred initially on stimulating the platelets with ADP. The results suggest that: (1) myosin is incorporated into the cytoskeleton transiently during shape change; (2) ADP interaction with the P(2T) receptor leads to incorporation of α-actinin and actin into the cytoskeleton as well as platelet aggregation; (3) further incorporation of α-actinin and myosin and incorporation of ABP and the 66K protein occur consequent to fibrinogen binding and platelet aggregation; (4) displacement of the 31K protein from the cytoskeleton is also a consequence of fibrinogen binding and platelet aggregation; (5) platelet disaggregation is accompanied by reversal of any cytoskeletal changes that have already occurred; (6) continuous occupation of the P(2T) receptor is required for maintenance of the cytoskeletal changes; (7) CAMP inhibits and reverses cytoskeletal assembly; and (8) MgCl(2) acts similarly to a GpIIb/IIIa antagonist under these experimental conditions.

Effects of ticlopidine administered to healthy volunteers on platelet function in whole blood.

Ticlopidine is thought to be a selective inhibitor of ADP-induced platelet function. Here we have investigated the effects of ticlopidine on platelet function in whole blood induced by ADP and by other platelet agonists. Whole blood was used because it was considered that ADP derived from red cells might act synergistically with other platelet agonists to enhance platelet responses, and that ticlopidine might interfere with this process. Measurements were performed using blood from 16 healthy volunteers before ticlopidine administration, after taking ticlopidine 250 mg daily for 10 days, after taking ticlopidine 250 mg twice daily for a further 10 days, and after 14 days off treatment. Ticlopidine proved to be a very effective inhibitor of the platelet aggregation induced by ADP; it was most effective in enhancing the reversibility of the aggregation response. The drug modestly but significantly reduced streptokinase, adrenaline, collagen, sodium arachidonate, PAF and U46619 - induced platelet aggregation. The drug significantly reduced the extent of the release reaction (14C-5HT release) induced by ADP, streptokinase, PAF, ristocetin and sodium arachidonate, and also reduced the extent of the synergistic 14C-5HT release induced by combinations of ADP and PAF, ADP and adrenaline and PAF and adrenaline. The various inhibitory effects of ticlopidine were evident after treatment with 250 mg daily but were more pronounced after 250 mg twice daily. All values had returned to normal after 14 days off treatment. Ticlopidine had no effect on serum thromboxane B2 production nor on several parameters of coagulation and fibrinolysis. We conclude that ticlopidine is an effective inhibitor of ADP-induced platelet aggregation and also the platelet aggregation and 14C-5HT release induced in whole blood by a number of platelet agonists and combinations of agonists. These latter effects are probably mainly via a selective effect on ADP. The inhibitory effects of the drug are dose-related.

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)

Inhibition of platelet aggregation by transdermal glyceryl trinitrate.

OBJECTIVE: To determine the optimum conditions for the demonstration of an antiplatelet effect of nitric oxide and to use these conditions to elucidate the effects of a transdermal glyceryl trinitrate patch on platelet aggregation in normal volunteers. METHODS: An open prospective crossover study. The effects of nitric oxide on platelet aggregation in whole blood and platelet rich plasma as stimulated by adenosine diphosphate and U46619 was assessed in the presence and absence of iloprost and MB22948. Optimum conditions for the demonstration of an antiplatelet effect of nitric oxide were then applied to whole blood from normal volunteers in the presence and absence of a transdermal glyceryl trinitrate patch. SETTING: University hospital. SUBJECTS: Eight normal volunteers. MAIN OUTCOME MEASURES: Platelet aggregation in the presence and absence of transdermal glyceryl trinitrate. RESULTS: The optimum conditions for the demonstration of an antiplatelet effect of nitric oxide in whole blood were collecting blood into a tube containing MB22948 and citrate and stimulating platelet aggregation with adenosine diphosphate in the presence or absence of iloprost. Using this method a significant effect of transdermal glyceryl trinitrate on platelet aggregation was shown (P < 0.03) in the presence and absence of iloprost. CONCLUSIONS: These results are consistent with an inhibitory effect on platelet aggregation of nitric oxide liberated by transdermal glyceryl trinitrate. Optimum test conditions are needed to show this effect. The clinical significance of the antiplatelet effect of transdermal glyceryl trinitrate is unknown.

Identification of a glycosidase activity with apparent specificity for 2-deoxy-D-glucose in glycosidic linkage.

2-Deoxy-D-glucose (dGlc) is a carbohydrate with significant activity as an inhibitor of glucose metabolism and as a precursor in the synthesis of glycosylated macromolecules; several of the enzymes associated with its metabolism remain uncharacterized. In the present report, the partial purification and some of the properties of a mammalian enzyme that appears to be relatively specific for the hydrolysis of dGlc bound in glycosidic linkage is described. The physiological function of this enzymatic activity is unknown. In addition, dGlc has been shown to be taken up by HTC cells in culture and incorporated into macromolecular bound form, both as dGlc and as 2-deoxygalactose which is formed from dGlc.

Relationship of spontaneous chemical transformation of arylsulfonylhydrazones of 2-pyridinecarboxaldehyde 1-oxide to anticancer activity.

The arylsulfonyl-hydrazones of 2-pyridinecarboxaldehyde 1-oxide represent a relatively new class of antineoplastic agents with the potential for clinical usefulness. The requirement for spontaneous chemical transformation of these agents to exert anticancer activity was evaluated using as the prototype the most potent member of this class synthesized to date, the 3,4-dimethoxybenzene sulfonylhydrazone of 2-pyridinecarboxaldehyde 1-oxide (3,4-DSP. 3,4-DSP was chemically unstable, decomposing with a half-life of 19 min in 0.01 M potassium phosphate buffer (pH 7.4) at 37 degrees. The major chemical decomposition product was identified as 2-pyridylcarbinol 1-oxide by comparison with the authentic compound. This carbinol is hypothesized to be formed via the intramolecular abstraction of hydrogen from the arylsulfonyl-hydrazone, a process that leads to the release of 3,4-dimethoxybenzenesulfinic acid and the formation of 1-oxidopyridin-2-yldiazomethane, which subsequently reacts with water. The diazomethane intermediate is a potent alkylating agent which, if generated in cells, would have the potential to alkylate nucleophilic groups of biologically important macromolecules. The proposed reactive species was trapped using both 4-(4-nitrobenzyl)pyridine (NBP) and morpholine, and the latter product was characterized by mass spectroscopy. The importance of the chemical formation of an alkylating species to cytotoxicity was demonstrated by studies in which solutions of 3,4-DSP were "aged" prior to addition to L1210 leukemia cells in culture and prior to incubation with NBP. The "aging" of 3,4-DSP for 20 min resulted in a 4-fold decrease in cytotoxicity, and aging for 1 to 3 hr led to complete loss of cytotoxicity. Correspondingly, a 20-min aging period decreased alkylation of NBP by 51%, and 3-hr aging resulted in essentially no alkylation of the nucleophile. Further support for the above proposed chemical activation pathway was provided by correlations between in vitro cytotoxicity, in vivo antineoplastic activity, chemical stability, and the degree of alkylation of NBP by a wide variety of arylsulfonyl-hydrazones. The lack of the 1-oxide, envisioned to be required for intramolecular hydrogen abstraction, the steric prevention of the abstraction, or the replacement of the proton of the nitrogrn of the side-chain by a methyl group resulted in a marked increase in chemical stability and a corresponding loss of the ability to alkylate NBP and to inhibit the replication of L1210 leukemia cells in culture.

Synthesis and biological activity of potential antimetabolites of L-fucose.

6-Substituted 6-deoxy-L-galactose (L-fucose) derivatives were synthesized as potential antimetabolites of L-fucose. The cytotoxic effects of these compounds were evaluated on P388 leukemia cells in culture. The L-fucose analogues which showed the most potent growth inhibition were the sulfonyl ester, bromo, and iodo derivatives; since these compounds were all capable of alkylation, it is conceivable that their cytotoxic action is a consequence of this property. In agreement with this interpretation, none of the agents synthesized showed specific inhibition of the incorporation of L-[3H]fucose into glycoprotein.