The Antidiabetic Drug Liraglutide Minimizes the Non-Cholinergic Neurotoxicity of the Pesticide Mipafox in SH-SY5Y Cells.

Organophosphorus (OPs) compounds have been widely used in agriculture, industry, and household, and the neurotoxicity induced by them is still a cause of concern. The main toxic mechanism of OPs is the inhibition of acetylcholinesterase (AChE); however, the delayed neuropathy induced by OPs (OPIDN) is mediated by other mechanisms such as the irreversible inhibition of 70% of NTE activity (neuropathy target esterase) that leads to axonal degeneration. Liraglutide is a long-lasting GLP-1 analog clinically used as antidiabetic. Its neurotrophic and neuroprotective effects have been demonstrated in vitro and in experimental models of neurodegenerative diseases. As in OPIDN, axonal degeneration also plays a role in neurodegenerative diseases. Therefore, this study investigated the protective potential of liraglutide against the neurotoxicity of OPs by using mipafox as a neuropathic agent (at a concentration able to inhibit and age 70% of NTE activity) and a neuronal model with SH-SY5Y neuroblastoma cells, which express both esterases. Liraglutide protected cells against the neurotoxicity of mipafox by increasing neuritogenesis, the uptake of glucose, the levels of cytoskeleton proteins, and synaptic-plasticity modulators, besides decreasing the pro-inflammatory cytokine interleukin 1ß and caspase-3 activity. This is the first study to suggest that liraglutide might induce beneficial effects against the delayed, non-cholinergic neurotoxicity of OPs.

Hydrolyzing activities of phenyl valerate sensitive to organophosphorus compounds paraoxon and mipafox in human neuroblastoma SH-SY5Y cells.

The molecular targets of best known neurotoxic effects associated to acute exposure to organophosphorus compounds (OPs) are serine esterases located in the nervous system, although there are other less known neurotoxic adverse effects associated with chronic exposure to OPs whose toxicity targets are still not identified. In this work we studied sensitivity to the non-neuropathic OP paraoxon and to the neuropathic OP mipafox of phenyl valerate esterases (PVases) in intact and lysed human neuroblastoma SH-SY5Y cells. The main objective was to discriminate different unknown pools of esterases that might be potential targets of chronic effects from those esterases already known and recognized as targets to these acute neurotoxicity effects. Two components of PVases of different sensitivities were discriminated for paraoxon in both intact and lysed cells; while the two components inhibitable by mipafox were found only for intact cells. A completely resistant component to paraoxon of around 30% was found in both intact and lysed cells; while a component of slightly lower amplitude (around 20%) completely resistant to mipafox was also found for both preparations (intact and lysed cells). The comparison of the results between the intact cells and the lysed cells suggests that the plasma membrane could act as a barrier that reduced the bioavailability of mipafox to PVases. This would imply that the discrimination of the different esterases should be made in lysed cells. However, those studies which aim to determine the physiological role of these esterases should be necessarily conducted in intact cultured cells.

L- and T-type calcium channel blockers protect against the inhibitory effects of mipafox on neurite outgrowth and plasticity-related proteins in SH-SY5Y cells.

Some organophosphorus compounds (OP), including the pesticide mipafox, produce late onset distal axonal degeneration, known as organophosphorus-induced delayed neuropathy (OPIDN). The underlying mechanism involves irreversible inhibition of neuropathy target esterase (NTE) activity, elevated intracellular calcium levels, increased activity of calcium-activated proteases and impaired neuritogenesis. Voltage-gated calcium channels (VGCC) appear to play a role in several neurologic disorders, including OPIDN. Therefore, this study aimed to examine and compare the neuroprotective effects of T-type (amiloride) and L-type (nimodipine) VGCC blockers induced by the inhibitory actions of mipafox on neurite outgrowth and axonal proteins of retinoic-acid-stimulated SH-SY5Y human neuroblastoma cells, a neuronal model widely employed to determine the neurotoxicity attributed to OP. Both nimodipine and amiloride significantly blocked augmentation of intracellular calcium levels and activity of calpains, as well as decreased neurite length, number of differentiated cells, and lowered concentrations of growth-associated protein 43 (GAP-43) and synapsin induced by mipafox. Only nimodipine inhibited reduction of synaptophysin levels produced by mipafox. These findings demonstrate a role for calcium and VGCC in the impairment of neuronal plasticity mediated by mipafox. Data also demonstrated the neuroprotective potential of T-type and L-type VGCC blockers to inhibit OP-mediated actions, which may be beneficial to counteract cases of pesticide poisoning.

High concentration of trichlorfon (1mM) disrupts axonal cytoskeleton and decreases the expression of plasticity-related proteins in SH-SY5Y cells.

Some organophosphorus compounds (OPs) induce a neurodegenerative disorder known as organophosphate-induced delayed neuropathy (OPIDN), which is related to irreversible inhibition of neuropathy target esterase (NTE) and impairment of neurite outgrowth. The present study addresses the effects of trichlorfon, mipafox (neuropathic model) and paraoxon (non-neuropathic model) on neurite outgrowth and neuroplasticity-related proteins in retinoic-acid-stimulated SH-SY5Y cells, a cellular model widely used to study the neurotoxicity of OPs. Mipafox (20µM) decreased cellular differentiation and the expression of neurofilament 200 (NF-200), growth associated- (GAP-43) and synaptic proteins (synapsin I and synaptophysin); whereas paraoxon (300µM) induced no effect on cellular differentiation, but significant decrease of NF-200, GAP-43, synapsin I and synaptophysin as compared to controls. However, the effects of paraoxon on these proteins were significantly lower than the effects of mipafox. In conclusion, axonal cytoskeletal proteins, as well as axonal plasticity-related proteins are more effectively affected by neuropathic (mipafox) than by non-neuropathic (paraoxon) OPs, suggesting that they might play a role in the mechanism of OPIDN. At high concentration (1mM), trichlorfon induced effects similar to those of the neuropathic OP, mipafox (20µM), but also caused high inhibition of AChE. Therefore, these effects are unlikely to occur in humans at non-lethal doses of trichlorfon.

Genomic and phenotypic alterations of the neuronal-like cells derived from human embryonal carcinoma stem cells (NT2) caused by exposure to organophosphorus compounds paraoxon and mipafox.

Historically, only few chemicals have been identified as neurodevelopmental toxicants, however, concern remains, and has recently increased, based upon the association between chemical exposures and increased developmental disorders. Diminution in motor speed and latency has been reported in preschool children from agricultural communities. Organophosphorus compounds (OPs) are pesticides due to their acute insecticidal effects mediated by the inhibition of acetylcholinesterase, although other esterases as neuropathy target esterase (NTE) can also be inhibited. Other neurological and neurodevelopmental toxic effects with unknown targets have been reported after chronic exposure to OPs in vivo. We studied the initial stages of retinoic acid acid-triggered differentiation of pluripotent cells towards neural progenitors derived from human embryonal carcinoma stem cells to determine if neuropathic OP, mipafox, and non-neuropathic OP, paraoxon, are able to alter differentiation of neural precursor cells in vitro. Exposure to 1 µM paraoxon (non-cytotoxic concentrations) altered the expression of different genes involved in signaling pathways related to chromatin assembly and nucleosome integrity. Conversely, exposure to 5 µM mipafox, a known inhibitor of NTE activity, showed no significant changes on gene expression. We conclude that 1 µM paraoxon could affect the initial stage of in vitro neurodifferentiation possibly due to a teratogenic effect, while the absence of transcriptional alterations by mipafox exposure did not allow us to conclude a possible effect on neurodifferentiation pathways at the tested concentration.

Over-expression of neuropathy target esterase activity in bovine chromaffin cell cultures by adenovirus-mediated gene transfer.

Chromaffin cells in culture show high neuropathy target esterase (NTE) activity. It is well known that inhibition and specific modification of NTE by some organophosphorus (OPs) compounds induces a neurodegenerative neuropathy. It has been suggested that NTE is responsible for phosphatidylcholine homeostasis, although its role in neuropathy induction remains unclear. The cDNA of human NTE (4.4kbp) was inserted into an adenoviral vector. Bovine chromaffin cells cultured at 50,000 cells/well were incubated with the vector for 2h and after removing the volume of infection, cells were maintained in the incubator. After 24h, NTE activity was 6.8+/-0.5mU/10(6) cells in untreated cells and 14.8+/-1.5mU/10(6) cells, 19.3+/-2.9mU/10(6) cells, 24.8+/-0.9mU/10(6) cells and 30.9+/-1.0mU/10(6) cells in cells incubated with 2, 4, 8 and 16microl of vector, respectively. After 60min of inhibition with mipafox increased concentrations, the calculated I(50) (60min) values were 5.5, 6.2 and 6.6microM for cells infected with 0, 2 and 10microl of vector preparation. We confirm that the adenoviral vector containing the human NTE gene is active in bovine chromaffin cells in culture and that the NTE activity expressed by the vector shows the same inhibition pattern by the neuropathic OP mipafox as the NTE activity of bovine chromaffin cells and cells remained viable after the high NTE activity expression.

Degradation of organophosphorus neurotoxicity in SY5Y neuroblastoma cells by organophosphorus hydrolase (OPH).

Numerous approaches have been studied to degrade organophosphorus (OP) compounds and ameliorate their toxicity. In the current study, the potential of genetically engineered organophosphorus hydrolase (OPH) enzymes to functionally biotransform OP neurotoxicants was examined by assessing effects of OPH-hydrolyzed OPs on acute and delayed indicators of neurotoxicity. SY5Y human neuroblastoma cells were used as a model test system, as these cells respond distinctly to mipafox, which produces OP-induced delayed neuropathy, and paraoxon, which does not. Short-term effects of four OPH-treated OPs on acetylcholinesterase (AChE) and neuropathy target esterase (NTE) activities were measured in retinoic acid-differentiated or undifferentiated cells, and delayed effects of OPH-treated paraoxon or mipafox on levels of neuronal cytoskeletal proteins in nerve growth factor (NGF)-differentiated cells. The anti-AChE activity of paraoxon (maximum 3 muM) and anti-NTE activity of mipafox (250 muM) in SY5Y cells were prevented by biodegradation with OPH. Anti-AChE activities of mipafox, methyl parathion, and demeton-S were partially ameliorated, depending on OP concentration. Intracellular amounts of the 200-kD neurofilament protein NF200 were unchanged after treatment with OPH-treated or buffer-treated paraoxon, as expected, as this endpoint is insensitive to paraoxon. However, NF200 levels rose in cells treated during late differentiation with OPH-treated mipafox. This finding suggests the existence of a threshold concentration of mipafox below which SY5Y cells can maintain their viability for compensating cellular damage due to mipafox in neurite elongation. These results indicate that OPH may be used to biodegrade OPs and remediate their neurotoxic effects in vitro and that AChE and NTE are suitable detectors for OPH amelioration.

Reduction of neuropathy target esterase does not affect neuronal differentiation, but moderate expression induces neuronal differentiation in human neuroblastoma (SK-N-SH) cell line.

Neuropathy target esterase (NTE) is inhibited and aged by organophosphorus compounds that induce delayed neuropathy in human and some sensitive animals. NTE has been proposed to play a role in neurite outgrowth and process elongation during neurodifferentiation. However, to date, there is no direct evidence of the relevance of NTE in neurodifferentiation under physiological conditions. In this study, we have investigated a possible role for NTE in the all-trans retinoic acid-induced differentiation of neuroblastoma cells. The functional inactivation of NTE by RNA interference indicated that reduction of NTE does not affect process outgrowth or differentiation of the cells, although moderate expression of NTE by expression of the NTE esterase domain accelerates the elongation of neurite processes. Mipafox, a neurotoxic organophosphate, was shown to block process outgrowth and differentiation in cells that have lowered NTE activity due to RNA interference, suggesting that mipafox may interact with other molecules to exert its effect in this context.

Effects of organophosphorus compounds on ATP production and mitochondrial integrity in cultured cells.

Recent studies in vivo and in vitro suggested that mitochondrial dysfunction follows exposure to organophosphorus (OP) esters. As mitochondrial ATP production is important for cellular integrity, ATP production in the presence of OP neurotoxicants was examined in a human neuronal cell line (SH-SY5Y neuroblastoma cells) and primary dorsal root ganglia (DRG) cells isolated from chick embryos and subsequently cultured to achieve maturation with axons. These cell culture systems were chosen to evaluate toxic effects on the mitochondrial respiratory chain associated with exposure to OP compounds that do and do not cause OP-induced delayed neuropathy (OPIDN), a disorder preceded by inhibition of neurotoxic esterase (NTE). Concentration- and time-response studies were done in neuroblastoma cells exposed to phenyl saligenin phosphate (PSP) and mipafox, both compounds that readily induce delayed neuropathy in hens, or paraoxon, which does not. Phenylmethylsulfonyl fluoride (PMSF) was included as a non-neuropathic inhibitor of NTE. Purified neuronal cultures from 9 day-old chick embryo DRG were treated for 12 h with 1 microM PSP, mipafox, or paraoxon. In situ evaluation of ATP production measured by bioluminescence assay demonstrated decreased ATP concentrations both in neuroblastoma cells and chick DRG neurons treated with PSP. Mipafox decreased ATP production in DRG but not in SH-SY5Y cells. This low energy state was present at several levels of the mitochondrial respiratory chain, including Complexes I, II, III, and IV, although Complex I was the most severely affected. Paraoxon and PMSF were not effective at all complexes, and, when effective, required higher concentrations than needed for PSP. Results suggest that mitochondria are an important early target for OP compounds, with exposure resulting in depletion of ATP production. The targeting of neuronal, rather than Schwann cell mitochondria in DRG following exposure to PSP and mipafox was verified by loss of the mitochondrial-specific dye, tetramethylrhodamine, in these cells. No such loss was seen in paraoxon exposed neurons isolated from DRG or in Schwann cells treated with any of the test compounds.

Neurofilament 200 as an indicator of differences between mipafox and paraoxon sensitivity in Sy5Y neuroblastoma cells.

Organophosphorus (OP) compounds produce potent neurotoxic effects in humans, including organophosphorus-induced delayed neuropathy (OPIDN). This investigation examined the potential for the 200-kD neurofilament protein (NF200) and other neuronal proteins to serve as indicators for neurite damage in a differentiated SY5Y human neuroblastoma cell culture system. Mipafox, which induces OPIDN, increased NF200 protein expression in SY5Y cells differentiated with human recombinant beta-nerve growth factor (NGF, 20 ng/ml) in a concentration-dependent manner, compared to NGF controls, when SY5Y cells were exposed to 0.3 or 30 microM mipafox during the last 5 days of neurite extension (experimental set A). However, mipafox produced little change in NF200 protein expression in SY5Y cells exposed continuously throughout neurite elongation (experimental set B). Paraoxon (up to 30 microM), which does not produce OPIDN, did not produce any change in NF200 expression in set A or set B. The upregulation of NF200 by mipafox may represent a compensatory response to neurite degeneration. Two other neuronal proteins, growth-associated protein 43 (GAP43) and microtubule-associated protein 2ab (MAP2ab), showed no changes in response to OP treatment in NGF-treated cells. Protein expression of NF200 was shown to be an indicator by which the sensitivities of SY5Y cells to mipafox and paraoxon were distinguishable at the molecular level. These results indicate an alternative approach and test system for investigating structure-activity relationships of OPs.

Neurotoxicity of polycyclic aromatic hydrocarbons and simple chemical mixtures.

Polycyclic aromatic hydrocarbons (PAHs) are a major class of environmental pollutants. These chemicals are the products of incomplete combustion and are present in every compartment of the environment. While the carcinogenic potential of these chemicals has been investigated in numerous studies, very little is known about the potential of these chemicals to produce damage to neural cells. The objective of this study was to investigate the toxicity of several model PAHs and binary mixtures of these chemicals in neural cells. Chemicals tested included benzo[a]pyrene (BaP), chrysene, anthracene, and pentachlorophenol (PCP). Four end points, including amino acid incorporation, total protein, total cell count, and viable cells (trypan dye exclusion), were measured in SY5Y human neuroblastoma cells and C6 rat glioma cells. The most sensitive measure of PAH toxicity in neural cells was amino acid incorporation into proteins. BaP was the most toxic of all PAHs tested, and anthracene failed to produce a toxic response at any concentration tested. Without metabolic activation, BaP induced a significant cytotoxic response at a concentration of 30 microM. With activation (0.25% S9), BaP induced a response at concentration levels of 3 microM and 30 microM. Minimal toxicity was observed with chrysene at the highest concentration tested, and anthracene failed to produce a toxic response at any concentration tested. With mixtures of PAHs the majority of samples induced additive responses. The minimum concentration required to induce a significant response was reduced for the mixture of chrysene and BaP when compared to BaP alone. In addition, PCP appeared to increase the inhibition of acetylcholinesterase by mipafox. The data suggest that PAHs are capable of producing damage to neural cells only at concentrations that are near their solubility limits.

Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds.

Organophosphorus (OP) compounds used as insecticides and chemical warfare agents are known to cause potent neurotoxic effects in humans and animals. Organophosphorus-induced delayed neuropathy (OPIDN) is currently thought to result from inhibition of neurotoxic esterase (NTE), but the actual molecular and cellular events leading to the development of OPIDN have not been characterized. This investigation examined the effects of OP compounds on the SY5Y human neuroblastoma cells at the cellular level to further characterize cellular targets of OP neurotoxicity. Mipafox and paraoxon were used as OP models that respectively do and do not induce OPIDN. Mipafox (0.05 mM) significantly decreased neurite length in SY5Y cells differentiated with nerve growth factor (NGF) while paraoxon at the same concentration had no effect when evaluated after each of three 4-day developmental windows during which cells were treated daily with OP or vehicle. In contrast, paraoxon but not mipafox altered intracellular calcium ion levels ([Ca(2+)](i)), as seen in three types of experiments. First, immediately following the addition of a single high concentration of OP to the culture, paraoxon caused a transient increase in [Ca(2+)](i), while mipafox up to 2 mM had no effect. Paraoxon hydrolysis products could also increase intracellular Ca(2+) levels, although the pattern of rise was different than it appeared immediately after paraoxon administration. Second, repeated low-level paraoxon treatment (0.05 mM/day for 4 days) decreased basal [Ca(2+)](i) in NGF-differentiated cells, though mipafox had no effect. Third, carbachol, a muscarinic acetylcholine receptor agonist, transiently increased [Ca(2+)](i) in differentiated cells, an affect attenuated by 4-day pretreatment with paraoxon (0.05 mM/day), but not by pretreatment with mipafox. These results indicate that the decrease in neurite extension that resulted from mipafox treatment was not caused by a disruption of Ca(2+) homeostasis. The effects of OPs that cause or do not cause OPIDN were clearly distinguishable, not only by their effects on neurite length, but also by their effects on Ca(2+) homeostasis in differentiated SY5Y cells.

Organophosphorus neuropathy target esterase inhibitors selectively block outgrowth of neurite-like and cell processes in cultured cells.

This study compares two direct-acting neuropathy target esterase (NTE) inhibitors (mipafox and 2-octyl-4H-1,3,2-benzodioxophosphorin 2-oxide (OBDPO)), a metabolic precursor to an NTE inhibitor (tri-o-cresyl phosphate or TOCP) and a potent acetylcholinesterase inhibitor (chlorpyrifos oxon or CPO) for their effects on outgrowth of neurite-like and cell processes and on viability in differentiated cultured cells (rat adrenal pheochromocytoma (PC-12) and brain glial tumor (C6)). The direct-acting NTE inhibitors block process outgrowth by 50% or more at 50-100 microM for OBDPO and 100-200 microM for mipafox, well below their cytotoxic levels (EC50 values, 445-474 microM for OBDPO and 1021-1613 microM for mipafox). In contrast, the effects on process development for TOCP and CPO parallel their cytotoxicity. These findings suggest that inhibition of neurite-like and cell process outgrowth by OBDPO and mipafox may be associated with NTE inhibition.

Interaction of organophosphorus compounds with muscarinic receptors in SH-SY5Y human neuroblastoma cells.

Human neuroblastoma cells (line SH-SY5Y) were used to examine the interaction of single exposure to organophosphorus compounds (OPs) with muscarinic receptors. In this study, SH-SY5Y cells were exposed for 30 min to concentrations of paraoxon, diisopropyl phosphorofluoridate (DFP), phenyl saligenin cyclic phosphate (PSP), and mipafox (N,N'-diisopropyl phosphorodiamide fluoridate) that ranged between 10(-9) M and 10(-3) M (10(-2) M for mipafox). Ability to interfere with muscarinic receptor binding was determined by change in the binding of the nonspecific antagonist [3H]-N-methylscopolamine (3H-NMS). Concentrations of paraoxon > 0.5 x 10(-3) M and PSP 1 x 10(-3) M significantly inhibited the binding of a saturating concentration of 3H-NMS. Concentrations of > 10(-5) M paraoxon or PSP could significantly inhibit the binding of a half-saturating concentration of 3H-NMS. Studies using specific antagonists for muscarinic subtypes (pirenzepine for M1, AFDX-116 for M2, and 4-DAMP for M3) indicated that SH-SY5Y cells have muscarinic receptors most sensitive to the specific antagonist for the M3 subtype (IC50 of 10(-8) M for 4-DAMP compared to 2.5 x 10(-6) M and 2.7 x 10(-5) M for pirenzepine and AFDX-116, respectively). As M3 receptor stimulation results in formation of inositol phosphates from membrane phosphoinositides the capability of OPs to alter levels of inositol phosphates and agonist-stimulated increases in inositol phosphate formation was examined. Intact cells were prelabeled with [3H]myo-inositol and then incubated for 15 min with the OPs before addition of 10(-5) M to 10(-3) M carbachol. Levels of inositol phosphates were determined as the amount of aqueous soluble radiolabeled product extracted from the reaction mixture. Paraoxon and PSP, but not mipafox or DFP, decreased basal levels of inositol phosphates in a concentration-related manner. This could be overcome in cells stimulated with carbachol, a muscarinic agonist, and with sodium fluoride, which does not act at muscarinic receptors. These results indicate that certain OPs, upon acute exposure, interact with muscarinic receptors, but that they also have effects on levels of inositol phosphates that may be associated with another site of action in SH-SY5Y cells.

Modification of mipafox-induced inhibition of neuropathy target esterase in neuroblastoma cells of human origin.

A neuroblastoma cell line of human origin was used as an in vitro model system to examine early effects on inhibition of neuropathy target esterase (NTE, also known as neurotoxic esterase) in the presence of agents belonging to classes of chemicals previously demonstrated to modify organophosphorus-induced delayed neuropathy in hens. For this study, differentiated SY-5Y cells were treated for up to 10 min with mipafox, an organophosphorus compound, and NTE inhibition was determined when cells exposed to mipafox were also exposed to the carbamate, aldicarb, and to the calcium channel blocker, verapamil. Cells were exposed to aldicarb or verapamil 5 min before, at the same time, or 2 min after mipafox. Less NTE inhibition was observed when either aldicarb or verapamil was included in the incubation of SY-5Y cells with mipafox. Effects of aldicarb and verapamil on NTE inhibition in differentiated SY-5Y cells were similar to effects in chicken brain homogenates. These results indicate that NTE inhibition can be detected in neuroblastoma cells, that these cells respond in a manner similar to chicken brain, and that mipafox-induced inhibition of NTE can be decreased in the presence of aldicarb or verapamil.

Effects of separate proteolytic and high-affinity binding activities of human thrombin on rapid platelet activation. A quenched-flow study.

We have used a general quenched-flow approach to study platelet function as early as 0.3 s after stimulation with three types of human thrombin: alpha-thrombin, gamma-thrombin, which is proteolytically active but does not bind to the high-affinity sites, and di-isopropyl fluorophosphate-derivatized (DIP)-alpha-thrombin, an active site-inhibited analogue that does bind to the high-affinity site. Large doses of gamma-thrombin evoked moderate aggregation and serotonin release, but minimal phosphorylation of the 20 and 47 kDa proteins. The initial (1.5-3.0 s) increase in cystolic free calcium concentration ([Ca2+]i) indicated by Indo-1 was also diminished, but by 5 s was nearly as high (1.0 microM) as with alpha-thrombin. A large dose of DIP-alpha-thrombin, on the other hand, induced minimal aggregation, serotonin secretion and [Ca2+]i response within 6 s. There was, however, a transient dephosphorylation of the 20 kDa protein. When combined, gamma- and DIP-alpha-thrombin were approximately additive in their ability to induce aggregation and serotonin secretion, but strongly synergistic in phosphorylating the 20 and 47 kDa proteins. The [Ca2+]i increase was not, however, enhanced over that induced by gamma-thrombin alone. These results demonstrate that phosphorylation of either the 20 or 47 kDa proteins is not correlated with [Ca2+]i dynamics and is neither required nor directly involved in platelet aggregation and secretion induced by thrombin. The high-affinity binding activity of thrombin is not necessary for rapid platelet Ca2+ influx, aggregation and serotonin release within the first critical seconds of activation.

[Effect of thrombin on macrophage and lymphocyte functional activity]. / Vliianie trombina na funktsional'nuiu aktivnost' makrofagov i limfotsitov.

It has been shown that thrombin induces blast transformation of lymphocytes, stimulates the phagocytic activity of macrophages and limits their migration.

Release of plasminogen activator from isolated perfused dog heart.

The release and the local activity of plasminogen activator (PA) were studied in isolated perfused dog hearts, without or with intimal injury induced by means of a balloon catheter inserted into the left anterior descending coronary artery (LAD). Thrombin but not DFP-thrombin induced a dose-dependent PA release in doses of 8 to 32 units. ADP 20 or 200 mumol but not ergonovine 20 or 200 micrograms induced a weak PA release. The local PA activity was much lower in the LAD at 1 or 4 weeks after this injury than in the intact LAD. However, the release of PA from the hearts after intimal injury was similar to findings in the intact hearts. We conclude from this study that thrombin plays an important role in regulating the coagulation-fibrinolysis system in endothelial cells and that changes in the properties of the endothelial cells may lead to initiation and enhancement of atherosclerosis.