Advanced crystallisation methods for small organic molecules.

Molecular materials based on small organic molecules often require advanced structural analysis, beyond the capability of spectroscopic techniques, to fully characterise them. In such cases, diffraction methods such as single crystal X-ray diffraction (SCXRD), are one of the most powerful tools available to researchers, providing molecular and structural elucidation at atomic level resolution, including absolute stereochemistry. However SCXRD, and related diffraction methods, are heavily dependent on the availability of suitable, high-quality crystals, thus crystallisation often becomes the major bottleneck in preparing samples. Following a summary of classical methods for the crystallisation of small organic molecules, this review will focus on a number of recently developed advanced methods for crystalline material sample preparation for SCXRD. This review will cover two main areas of modern small organic molecule crystallisation, namely the inclusion of molecules within host complexes (e.g., "crystalline sponge" and tetraaryladamantane based inclusion chaperones) and the use of high-throughput crystallisation, employing "under-oil" approaches (e.g., microbatch under-oil and ENaCt). Representative examples have been included for each technique, together with a discussion of their relative advantages and limitations to aid the reader in selecting the most appropriate technique to overcome a specific analytical challenge.

NMDA receptors mediate calcium-dependent, bidirectional changes in dendritic PICK1 clustering.

AMPA receptor (AMPAR) trafficking at CNS synapses is regulated by several receptor-binding proteins. One model of AMPAR endocytosis entails the cotargeting of the GluR2-interacting protein PICK1 and activated PKC to synapses. We demonstrate that NMDA receptor (NMDAR) activation mediates bidirectional changes in surface AMPARs through two additional forms of PICK1 redistribution. In neurons, NMDAR activation, which induces AMPAR endocytosis, increases endosomal PICK1 clustering. In contrast, stronger NMDAR activation rapidly reduces PICK1 clustering accompanied by decreases in PICK1/GluR2 association and increases in surface AMPAR levels. PICK1-siRNA similarly increases surface AMPARs and occludes the NMDAR-mediated effect, demonstrating the role of PICK1 in this process. Bidirectional NMDAR-mediated changes in PICK1 localization are determined by the magnitude of receptor-activated dendritic calcium signals. Our results show that PICK1 localization in dendrites is subject to multiple forms of regulation that contribute to surface AMPAR expression, likely by modulating the numbers of AMPARs maintained in intracellular compartments.

Synergistic outside-in regulation of platelet activation by GPIIb/IIIa ligand-induced conformation and oligomerization.

Full platelet activation with serotonin secretion and thromboxane A(2) (TxA(2)) formation induced by a low dose of thrombin receptor agonist peptide (TRAP) or high dose ADP requires platelet aggregation. This requirement can be replaced by pretreatment of platelets with a combination of reagents including: GPIIb/IIIa inhibitors yielding ligand-induced binding sites (LIBS), either arginine-glycine-aspartate-serine (RGDS) peptide or Ro 43-5054, cytochalasin to disrupt actin filaments and crosslinking by a GPIIb/IIIa mAb (pl-62). Crosslinking is required since Fab fragments of pl-62 do not support activation. Engagement of the Fc receptor by the mAb Fc domain is not required for pl-62 augmentation, since it is not blocked by the anti-Fc receptor mAb, IV-3. Another GPIIb/IIIa inhibitor, Ro 44-9883, not yielding LIBS epitopes, serves as a negative control and shows a requirement for LIBS in addition to crosslinking. Focal adhesion kinase tyrosine phosphorylation induced by TRAP is blocked by these GPIIb/IIIa antagonists, but restored by pl-62 crosslinking independent of LIBS induction. Tyrosine phosphorylation of a peptide comigrating with p38 MAP kinase is also inhibited by these antagonists and restored by pl-62 crosslinking. However, p38 MAP kinase activation by low dose TRAP is not affected by these aggregation inhibitors. Tyrosine phosphorylation of a 34-kDa phosphoprotein in the absence of aggregation or TxA(2) formation was uniquely augmented by Ro 43-5054 but not Ro 44-9883 under the above activation conditions.

Effect of retroviral transduction of canine microvascular endothelial cells on beta(1) integrin subunit expression and cell retention to PTFE grafts.

This study evaluated the effect of retroviral transduction on canine microvascular endothelial cell (CMVEC) detachment from fibrin glue coated expanded polytetrafluoroethylene (ePTFE) graft material. CMVEC were isolated from adipose tissue by fluorescent activated cell sorting (FACS). Three treatment groups were evaluated: G-I, transduced CMVEC, selected in antibiotic G418 for 10 days (n=5); G-II, CMVEC selected in G418 and recovered from selection for 4 days (n=5); and G-III, control group of naive CMVEC (n=6). (3)H-thymidine labeled endothelial cells were seeded on fibrin glue coated four-mm diameter PTFE. Grafts were exposed to physiologic shear stresses of 16 dyn/cm(2). Cell detachment was determined by (3)H-thymidine counts in the circuit effluent. beta(1) integrin subunit expression was measured by flow cytometry. After 2 hours of flow exposure, G-I and G-II demonstrated significantly greater cell detachment rates compared with the control seeded grafts. Median peak channel beta(1) integrin subunit value for G-III CMVEC was 2311+/-481.7 vs. 31.5+/-4.51 and 26.3+/-2.0 in the transduced cell groups (p=0.00043). Low beta(1) integrin expression correlated with flow induced high detachment rates of retrovirally-transduced CMVEC.

Role of AMPA receptor endocytosis in synaptic plasticity.

Activity-mediated changes in the strength of synaptic communication are important for the establishment of proper neuronal connections during development and for the experience-dependent modification of neural circuitry that is believed to underlie all forms of behavioural plasticity. Owing to the wide-ranging significance of synaptic plasticity, considerable efforts have been made to identify the mechanisms by which synaptic changes are triggered and expressed. New evidence indicates that one important expression mechanism of several long-lasting forms of synaptic plasticity might involve the physical transport of AMPA-type glutamate receptors in and out of the synaptic membrane. Here, we focus on the rapidly accumulating evidence that AMPA receptors undergo regulated endocytosis, which is important for long-term depression.

Regulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD.

The endocytosis of AMPA receptors is thought to be important in the expression of long-term depression (LTD) triggered by NMDA receptor activation. Although signaling pathways necessary for LTD induction have been identified, those responsible for the regulated internalization of AMPA receptors are unknown. Here we show that activation of NMDA receptors alone can trigger AMPA receptor endocytosis through calcium influx and activation of the calcium-dependent protein phosphatase calcineurin. A distinct signaling mechanism mediates the AMPA receptor endocytosis stimulated by insulin. These results demonstrate that although multiple signaling pathways can induce AMPA receptor internalization, NMDA receptor activation enhances AMPA receptor endocytosis via a signaling mechanism required for the induction of LTD.

Lotrafiban: an oral platelet glycoprotein IIb/IIIa blocker.

Platelets play a major role in thrombus formation, as well as in the pathogenesis of atherothrombosis. Inhibition of platelet function is now emphasised more than ever for prevention and treatment of almost all vascular diseases, since thrombosis is established as the key pathogenic event causing acute ischaemic coronary and cerebrovascular syndromes. Although acetylsalicylic acid (aspirin) has been shown to reduce the incidence of myocardial infarction and stroke, its effect is weak and more effective antithrombotic agents are required to manage patients at high-risk for recurrent vascular events. Platelet glycoprotein IIb/IIIa receptor (GPIIb/IIIa) blockade represents a significant advance in interventional cardiology and treatment of acute ischaemic syndromes. The past several years have seen the introduction of many platelet GPIIb/IIIa blockers into the clinical arena targeting the unique platelet GPIIb/IIIa receptor for the adhesive proteins, fibrinogen and von Willebrand Factor. Platelet GPIIb/IIIa blockers administered intravenously have proven efficacious in mitigating arterial thrombosis in acute coronary syndromes (unstable angina and non-ST-elevation myocardial infarction) and percutaneous coronary interventions (PCI) such as balloon dilatation and stent implantation. Currently, orally-active platelet GPIIb/IIIa blockers are being developed to provide additional benefits for primary and secondary prevention of thrombosis as chronic treatment, especially in high-risk patients. Lotrafiban (SmithKline Beecham) is a member of the latest generation of orally-active platelet GPIIb/IIIa blockers undergoing Phase III clinical trials to test the relative effectiveness versus other oral platelet inhibitors for ischaemic conditions including unstable angina, restenosis after PCI and stroke. Lotrafiban is converted from an esterified prodrug by plasma and liver esterases to a peptidomimetic of the arginine-glycine-aspartic acid amino acid sequence. This sequence itself mimics the binding site of fibrinogen and von Willebrand Factor to the platelet GPIIb/IIIa receptor. Preliminary results of the clinical trial APLAUD (antiplatelet useful dose) show that lotrafiban is clinically safe and well-tolerated in patients with recent myocardial infarction, unstable angina, transient ischaemic attack (TIA), or stroke when added to aspirin therapy. With lotrafiban, a worldwide large-scale Phase III clinical trial BRAVO (blockage of the GPIIb/IIIa receptor to avoid vascular occlusion) is currently underway. In general, GPIIb/IIIa blockade seems clinically very promising. A number of unresolved issues, however, remain to be elucidated.

Platelet-activating factor (PAF)-induced decreases in whole-body and skeletal muscle protein synthesis.

Platelet-activating factor (PAF) has been shown to reduce rat skeletal muscle amino acid uptake, which may restrict intracellular amino acid availability for protein synthesis and amino acid oxidation during endotoxemia. We investigated in rats the effect of PAF infusion on amino acid and protein metabolism by measuring (a) whole-body and tissue leucine kinetics; (b) plasma amino acid profile; and (c) muscle RNA activity (protein synthesis efficiency) and relative abundance of myofibrillar proteins. Fasted male Sprague-Dawley rats (250+/-20 g) were given a 4-h i.v. continuous infusion of L-(1-14C)-leucine to determine leucine kinetics during the infusion of PAF (2 microg/kg PAF as a priming i.v. bolus 1 h before a 4-h i.v. infusion of 2 microg/kg/h PAF) or vehicle. PAF infusion caused sustained hypotension, hyperglycemia, hematological alterations, and hyperlacticacidemia. Whole-body protein synthesis was decreased by 24% (P < 0.05) and leucine flux oxidized was increased by 23% (P < 0.05). Leucine flux was reduced, although not significantly (P = 0.07), in PAF-treated rats (n = 8) compared with controls (n = 8). PAF significantly decreased fractional protein synthesis in the rectus abdominus (33%), soleus (30%), and extensor digitorum longus (26%) muscles, but not in the liver. Plasma branched-chain amino acid levels decreased (approximately 30%, P < 0.05) in PAF-treated rats. Muscle RNA activity was 32% lower and myosin relative abundance declined whereas actin was unchanged in PAF-treated rats. PAF induced net protein catabolism as a result of elevated leucine oxidation at the expense of protein synthesis. PAF had the cumulative effects in the skeletal muscle of (a) attenuating amino acid uptake, (b) reducing protein synthesis efficiency, (c) decreasing fractional protein synthesis rate, and (d) decreasing myosin relative abundance. Thus, PAF may be an important mediator of decreased protein synthesis in skeletal muscle during endotoxic and septic shock.

Vascular smooth muscle cell apoptosis in aneurysmal, occlusive, and normal human aortas.

PURPOSE: Apoptosis is a physiologic mechanism of cell death that regulates mass and architecture in many tissues. Apoptosis has been described as a feature in human vascular atherosclerosis and large vessel structural integrity. We examined the extent of vascular smooth muscle cell (VSMC) apoptosis in aneurysmal, occlusive, and normal human aortic tissue. METHODS: Tissue samples of aneurysmal, occlusive, and normal human infrarenal aorta were evaluated. DNA fragmentation detection methods, immunohistochemistry, and DNA electrophoresis determined VSMC density, VSMC apoptosis, and apoptosis markers. Apoptotic cells and VSMC nuclei were counted with the use of computer-generated image analysis. Aortic subtypes were compared statistically by analysis of variance. RESULTS: Seventeen aneurysmal, ten occlusive, and five normal human aortas were evaluated. By alpha(1)-actin immunostaining, VSMC density was least in aneurysmal aortas (271.8 +/- 13.5 cells/high-power field [HPF]) compared with occlusive aorta (278.2 +/- 39.4 cells/HPF) and normal aortas (291.0 +/- 25.4 cells/HPF; P = not significant). Presence of apoptotic VSMCs was demonstrated by terminal deoxynucleotidyl transferase fragment end labeling and propidium iodide nuclear staining. VSMC apoptosis was greatest within aneurysmal aortas with 11.7 +/- 1.5 cells/HPF compared with occlusive aortas with 3.3 +/- 0. 8 cells/HPF (P <.05) and normal aortas with 3.75 +/- 4.6 cells/HPF (P <.05). Significant differences in apoptosis markers, p53 or bcl-2, could not be demonstrated by immunohistochemistry or DNA electrophoresis in aortic subtypes. CONCLUSION: Apoptosis of VSMCs is increased and VSMC density is decreased within the medial layer of aneurysmal aortic tissue. Structural degeneration of aortic tissue at the cellular level contributes to aneurysmal formation.

SR-90107 (Sanofi-Synthélabo).

SR-90107 is a synthetic pentasaccharide heparinoid Factor Xa antagonist and thrombokinase inhibitor in joint development by Sanofi-Synthelabo (formerly Sanofi) and Organon as a potential treatment and prophylaxis for deep vein thrombosis (DVT) and symptomatic pulmonary embolism following hip or knee surgery and as a potential treatment for coronary artery diseases [330073,359231]. The compound is in phase III clinical trials for the prevention of DVT and pulmonary embolism; phase III trials for the treatment of DVT and pulmonary embolism were expected to start in the first quarter of 2000 and phase IIb trials in cardiology indications are also underway. NDAs are planned to be submitted in Europe and the US in the third quarter of 2000 for the prevention of DVT and symptomatic pulmonary embolism, in 2002 for the treatment of DVT and pulmonary embolism and in 2004 for the treatment of coronary artery diseases [359231]. DVT AND PULMONARY EMBOLISM: The compound had entered phase III clinical trials by December 1998 for the prevention of thrombosis [320585]. By February 2000, four phase III trials in the prevention of DVT and pulmonary embolism following orthopaedic surgery were underway: the European PENTHIFRA trial, which involves 1707 patients with hip fracture; the US PENTATHLON trial, which involves 2200 patients undergoing hip replacements; the European EPHESUS trial, which involves 2200 patients undergoing hip replacements; and the US PENTAMAKS trial, which involves 1000 patients undergoing major knee surgery [359231]. Clinical data from these trials are expected to be available by June 2000 [359793]. By February 2000, preparations were also being made for two phase III trials of SR-90107 for the treatment of DVT and pulmonary embolism, both expected to be initiated in the first quarter of 2000; the MATISSE DVT trial, a double-blind trial of SR-90107 versus enoxaparin sodium in 2200 patients; and the MATISSE PE trial, an open study of SR-90107 versus unfractionated heparin in 2200 patients [359231]. CORONARY ARTERY DISEASES: By February 2000, SR-90107 was also under development for unstable angina, percutaneous transluminal coronary angioplasty, and acute myocardial infarction. At this time, the phase IIb PENTALYSE trial in thrombolyzed acute myocardial infarction patients had been completed, demonstrating a good safety/efficacy ratio, and the phase IIb PENTUA trial in unstable angina was ongoing [359231]. In October 1999, Merrill Lynch forecast sales of EUR 180 million in 2003, planning a review of this figure once clinical data were available [346209]. Also in October 1999, Lehman Brothers predicted that the product had a 70% chance of reaching the market with potential peak sales of US 700 million dollars in 2008 [346267].

Role of AMPA receptor cycling in synaptic transmission and plasticity.

Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF caused a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis caused a gradual increase in the AMPAR EPSC. These manipulations had no effect on the NMDAR EPSC but prevented the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process.

Dynamin-dependent endocytosis of ionotropic glutamate receptors.

Little is known about the mechanisms that regulate the number of ionotropic glutamate receptors present at excitatory synapses. Herein, we show that GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are removed from the postsynaptic plasma membrane of cultured hippocampal neurons by rapid, ligand-induced endocytosis. Although endocytosis of AMPARs can be induced by high concentrations of AMPA without concomitant activation of N-methyl-D-aspartate (NMDA) receptors (NMDARs), NMDAR activation is required for detectable endocytosis induced by synaptically released glutamate. Activated AMPARs colocalize with AP2, a marker of endocytic coated pits, and endocytosis of AMPARs is blocked by biochemical inhibition of clathrin-coated pit function or overexpression of a dominant-negative mutant form of dynamin. These results establish that ionotropic receptors are regulated by dynamin-dependent endocytosis and suggest an important role of endocytic membrane trafficking in the postsynaptic modulation of neurotransmission.

Rapid redistribution of glutamate receptors contributes to long-term depression in hippocampal cultures.

Synaptic strength can be altered by a variety of pre- or postsynaptic modifications. Here we test the hypothesis that long-term depression (LTD) involves a decrease in the number of glutamate receptors that are clustered at individual synapses in primary cultures of hippocampal neurons. Similar to a prominent form of LTD observed in hippocampal slices, LTD in hippocampal cultures required NMDA receptor activation and was accompanied by a decrease in the amplitude and frequency of miniature excitatory postsynaptic currents. Immunocytochemical analysis revealed that induction of LTD caused a concurrent decrease in the number of AMPA receptors clustered at synapses but had no effect on synaptic NMDA receptor clusters. These results suggest that a subtype-specific redistribution of synaptic glutamate receptors contributes to NMDA receptor-dependent LTD.

Rapid, activation-induced redistribution of ionotropic glutamate receptors in cultured hippocampal neurons.

We have examined the membrane localization of an AMPA receptor subunit (GluR1) and an NMDA receptor subunit (NR1) endogenously expressed in primary cultures of rat hippocampal neurons. In unstimulated cultures, both GluR1 and NR1 subunits were concentrated in SV2-positive synaptic clusters associated with dendritic shafts and spines. Within 5 min after the addition of 100 microM glutamate to the culture medium, a rapid and selective redistribution of GluR1 subunits away from a subset of synaptic sites was observed. This redistribution of GluR1 subunits was also induced by AMPA, did not require NMDA receptor activation, did not result from ligand-induced neurotoxicity, and was reversible after the removal of agonist. The activation-induced redistribution of GluR1 subunits was associated with a pronounced (approximately 50%) decrease in the frequency of miniature EPSCs, consistent with a role of GluR1 subunit redistribution in mediating rapid regulation of synaptic efficacy. We conclude that ionotropic glutamate receptors are regulated in native neurons by rapid, subtype-specific membrane trafficking, which may modulate synaptic transmission in response to physiological or pathophysiological activation.

Effects of PKA and PKC on miniature excitatory postsynaptic currents in CA1 pyramidal cells.

Protein kinases play an important role in controlling synaptic strength at excitatory synapses on CA1 pyramidal cells. We examined the effects of activating cAMP-dependent protein kinase or protein kinase C (PKC) on the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) with perforated patch recording techniques. Both forskolin and phorbol-12,13-dibutryate (PDBu) caused a large increase in mEPSC frequency, but only PDBu increased mEPSC amplitude, an effect that was not observed when standard whole cell recording was performed. These results support biochemical observations indicating that PKC, similar to calcium/calmodulin-dependent protein kinase II, has an important role in controlling synaptic strength via modulation of AMPA receptor function, potentially through the direct phosphorylation of the GluR1 subunit.

The m3 muscarinic acetylcholine receptor differentially regulates calcium influx and release through modulation of monovalent cation channels.

Several types of transmembrane receptors regulate cellular responses through the activation of phospholipase C-mediated Ca2+ release from intracellular stores. In non-excitable cells, the initial Ca2+ release is typically followed by a prolonged Ca2+ influx phase that is important for the regulation of several Ca2+-sensitive responses. Here we describe an agonist concentration-dependent mechanism by which m3 muscarinic acetylcholine receptors (mAChRs) differentially regulate the magnitude of the release and influx components of a Ca2+ response. In transfected Chinese hamster ovary cells expressing m3 mAChRs, doses of the muscarinic agonist carbachol ranging from 100 nM to 1 mM evoked Ca2+ release responses of increasing magnitude; maximal Ca2+ release was elicited by the highest carbachol concentration. In contrast, Ca2+ influx was maximal when m3 mAChRs were activated by moderate doses (1-10 microM) of carbachol, but substantially reduced at higher agonist concentrations. Manipulation of the membrane potential revealed that the carbachol-induced Ca2+ influx phase was diminished at depolarized potentials. Importantly, carbachol doses above 10 microM were found to couple m3 mAChRs to the activation of an inward, monovalent cation current resulting in depolarization of the cell membrane and a selective decrease in the influx, but not release, component of the Ca2+ response. These studies demonstrate, in one experimental system, a mechanism by which a single subtype of G-protein-coupled receptor can utilize the information encoded in the concentration of an agonist to generate distinct intracellular Ca2+ signals.

Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors.

Distinct subtypes of glutamate receptors often are colocalized at individual excitatory synapses in the mammalian brain yet appear to subserve distinct functions. To address whether neuronal activity may differentially regulate the surface expression at synapses of two specific subtypes of ionotropic glutamate receptors we epitope-tagged an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor subunit (GluR1) and an NMDA (N-methyl-D-aspartate) receptor subunit (NR1) on their extracellular termini and expressed these proteins in cultured hippocampal neurons using recombinant adenoviruses. Both receptor subtypes were appropriately targeted to the synaptic plasma membrane as defined by colocalization with the synaptic vesicle protein synaptophysin. Increasing activity in the network of cultured cells by prolonged blockade of inhibitory synapses with the gamma-aminobutyric acid type A receptor antagonist picrotoxin caused an activity-dependent and NMDA receptor-dependent decrease in surface expression of GluR1, but not NR1, at synapses. Consistent with this observation identical treatment of noninfected cultures decreased the contribution of endogenous AMPA receptors to synaptic currents relative to endogenous NMDA receptors. These results indicate that neuronal activity can differentially regulate the surface expression of AMPA and NMDA receptors at individual synapses.

Signaling pathways for tissue factor expression in lipopolysaccharide-stimulated bovine alveolar macrophages.

OBJECTIVE: To investigate receptor-mediated intracellular events in bovine alveolar macrophages (AM) stimulated by bacterial lipopolysaccharide (LPS), using tissue factor (TF) expression as the measurable functional endpoint. SAMPLE POPULATION: Pulmonary AM harvested from 1- to 4-month-old male Holstein calves. PROCEDURE: Alveolar macrophages, acquired by use of volume-controlled bronchopulmonary lavage, were treated with CD14 monoclonal antibody (20 microg/ml), pertussis toxin (300 ng/ml), or 1 of 3 known protein kinase C (PKC) inhibitors (10 microM chelerythrin, 100 microM H-7, or 50 nM staurosporin), then were stimulated with LPS alone (0.01, 0.10, 1.0, 10.0 microg/ml) or LPS (0.25, 0.5, 1.0 ng/ml) in combination with concentrated bovine serum fraction 2 (500 ng/ml). Tissue factor expression was quantified by use of a colorimetric assay. Changes in intracellular Ca2+ concentration and pH were monitored, using Ca2+- and pH-sensitive fluorescent dyes, with changes in fluorescent intensity after incubation with LPS measured by spectrophotometry. RESULTS: Treatment of AM with a CD14 monoclonal antibody caused profound inhibition of TF expression (P < 0.0001) after stimulation by LPS combined with bovine serum fraction 2. Pertussis toxin had a significant (P < 0.0319) inhibitory effect on TF expression when cells were stimulated by LPS alone. Treatment with all 3 PKC inhibitors caused marked reduction in TF expression of cells stimulated with LPS alone or with phorbol myristate acetate. Stimulation of cells by LPS failed to mobilize intracellular Ca2+ stores or to alter cytosolic pH. CONCLUSION: LPS combined with serum factors binds to CD14 on the surface of AM, and PKC is an important signaling kinase in the pathway utilized by LPS, resulting in enhanced TF expression; a pertussis toxin-sensitive G protein is involved in the signaling pathway utilized by LPS alone; and mobilization of Ca2+ does not have a role in the signal transduction pathway utilized by LPS nor does LPS affect cytosolic pH of AM.

Blocking platelet aggregation inhibits thromboxane A2 formation by low dose agonists but does not inhibit phosphorylation and activation of cytosolic phospholipase A2.

Inhibition of aggregation by Ro 44-9883, a potent and selective non-peptide GPIIb/IIIa antagonist, resulted in inhibition of serotonin secretion induced by weak agonists such as ADP or low doses of either thrombin receptor agonist peptide (TRAP) or collagen. In contrast, alpha granule secretion was inhibited to different extents dependent on donor, averaging 60% inhibition. Inhibition of serotonin secretion correlated with an inhibition of thromboxane A2 (TxA2) formation, both of which were overcome by higher doses of TRAP or collagen. Ro 44-9883 had no effect on the already reduced serotonin secretion and TxA2 formation in Glanzmann's thrombasthenic platelets. Restoration of serotonin secretion in the absence of aggregation requires both TxA2 and lysophosphatidic acid. In addition, Ro 44-9883 inhibition of TxA2 formation was not due to a lack of phospholipase A2 (PLA2) phosphorylation and activation as assayed in vitro. These results suggest that aggregation is required for weak or low dose agonist induced in vivo activity of PLA2, possibly by either regulating phospholipid substrate availability or interaction of PLA2 with platelet membranes.