Modulating effects of adenosine on the process of human platelet activation]

The inhibitory effect of adenosine on aggregation of human platelets activated by platelet activating factor (PAF), ADP and serotonin (5-HT) were examined using native platelets from blood of volunteers. Platelet aggregation was determined by Born's method. Effective adenosine concentrations (IC50) which had inhibited platelet aggregation were found to be 0.63 +/- 0.11, 1.47 +/- 0.31 and 0.64 +/- 0.18 microM, respectively. It was shown that 10 microM adenosine inhibited PAF-induced platelet aggregation completely. The same adenosine concentration blocked ADP- and 5-HT-induced aggregation only partially. Adenosine is physiological inhibitor of human platelet aggregation in administration of PAF, ADP and 5-HT. Specific characteristics of adenosine modulating effect on these ligands was elicited.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

The benzodiazepine receptor ligands RO 5-4864 and RO 15-1788 do not block the inhibition of PAF-induced platelet aggregation seen with the hetrazepine WEB2086

The hypothesis was tested that the hetrazepine WEB 2086 acts as an inhibitor of PAF-induced platelet aggregation via interaction with the platelet benzodiazepine receptor(BDZR). WEB 2086 is a potent inhibitor of rabbit platelet aggregation and ATP secretion induced by 370 nM PAF. The two BDZR ligands RO 5-4864 and RO 15-1788 (7-96 microM) are inactive as PAF antagonists. When platelets were pretreated with either BDZR ligand, and then exposed to various concentrations of WEB 2086, there was no alteration of the dose-response relationship of the hetrazepine on PAF-induced aggregation, as reflected by threshold concentration, ED50, or maximum inhibition seen with WEB 2086. Pretreatment of platelets with the BDZR ligands also failed to block the inhibitory action of WEB 2086 on PAF-induced ATP release. The data are consistent with the notion that WEB 2086 acts as a PAF antagonist through its action at a specific PAF receptor, and is dissociated from, and independent of, interaction with the benzodiazepine receptor.     

The differences in 12-hydroxyeicosatetraenoic acid and thromboxane B2 release from guinea pig platelets.

Anti-12(S)-hydroxyeicosatetraenoic acid (12-HETE)-antibody and anti-thromboxane B2 (TXB2)-antibody were generated and applied to the radioimmunoassay. The detection limit for 12-HETE was 16 pg. The cross-reactivities of anti-12-HETE-antibody were 4.6% for 15-HETE, 0.18% for 5-HETE and below 0.15% for leukotrienes and prostaglandins (PGs). 12-HETE and TXB2 released from guinea pig platelets were measured by radioimmunoassay. Platelet activating factor (PAF) at 10(-9) M induced the aggregation of platelets, the releases of immunoreactive-12-HETE (1.8 +/- 1.2 ng/10(8) platelets, mean +/- S.D.) and immunoreactive-TXB2 (18.5 +/- 17.3 ng/10(8) platelets). Collagen at 1 microgram/ml also evoked platelet aggregation, the releases of immunoreactive-12-HETE (2.7 +/- 1.1 ng/10(8) platelets) and immunoreactive-TXB2 (11.8 +/- 4.6 ng/10(8) platelets). By the stimulation with these compounds, TXB2 was produced in a greater amount than 12-HETE from guinea pig platelets. Although 10(-7) M and 10(-6) M U46619, a TXA2 mimetic, caused platelet aggregation, arachidonic acid metabolites were not released. These data suggest the presence of different mechanisms of platelet activation depending on each stimulus.     

P2X1-mediated ERK2 activation amplifies the collagen-induced platelet secretion by enhancing myosin light chain kinase activation

The ATP-gated P2X1 ion channel is the only P2X subtype expressed in human platelets. Via transmission electron microscopy, we found that P2X1 mediates fast, reversible platelet shape change, secretory granule centralization, and pseudopodia formation. In washed human platelets, the stable P2X1 agonist alpha,beta-methylene ATP (alpha,beta-meATP) causes rapid, transient (2-5 s), and dose-dependent myosin light chain (MLC) phosphorylation, requiring extracellular Ca2+. Phosphorylation was inhibited by the calmodulin (CaM) inhibitor W-7, but not by the Rho kinase inhibitor HA-1077, i.e. it is exclusively regulated by Ca2+/CaM-dependent MLC kinase. Correspondingly, the P2X1-induced platelet shape change was inhibited by W-7 and by the MLC kinase inhibitor ML-7 but not by HA-1077. W-7, ML-7, the protein kinase C inhibitor GF109203-X, and the Src family kinase inhibitor PP1 inhibited the collagen and convulxin-induced early platelet degranulation, shape change, and subsequent aggregation, indicating a role for Ca2+/CaM and MLC kinase in these glycoprotein VI-related platelet responses. The secreted ATP-mediated P2X1-dependent ERK2 activation induced by low collagen concentrations contributes to MLC kinase activation since P2X1 desensitization or blockade of ERK2 phosphorylation by U0126 strongly attenuated MLC phosphorylation, degranulation, and aggregation. We therefore conclude that at low doses of collagen, glycoprotein VI activation leads to early protein kinase C- and MLC kinase-dependent degranulation. Rapidly released ATP triggers P2X1 -mediated Ca2+ influx, activating ERK2, in turn amplifying platelet secretion by reinforcing the early MLC kinase phosphorylation. Hence, the P2X1-ERK2-MLC axis contributes to collagen-induced platelet activation by enhancing platelet degranulation.     

Role of Platelet-Actlvatlng-Factor (PAF) on Cellular Responses after Stimulation with Leptospire Lipopolysaccharide

Leptospire lipopolysaccharide (LPS) stimulated the adherence of polymorphonuclear neutrophils (PMNs) to human umbilical vein endothelial cells (HUVEC). Enhanced PMN adherence in response to leptospire LPS can be mediated by platelet-activator-factor (PAF), because a PAF antagonist reduced adherence. Leptospire LPS also induced the adherence platelets or U937. The second experiment involved leptospire LPS elicited platelet aggregation in a PMN-platelet mixture, because leptospire LPS stimulated human PMN but not the human platelets. The platelet response was observed only in the mixture system and was inhibited by a PAF antagonist. PAF could be an important pathogenic factor in human leptospirosis.     

Characterization of the effect of the adenosine agonist cyclohexyladenosine on platelet activating factor-induced increases in [Ca]i in human platelets in vitro

Previous studies have shown that adenosine agonists acting at A-2 receptors inhibit platelet aggregation. Since an increase in cytosolic Ca2+ concentration (delta [Ca2+]i) is closely associated with the time frame of platelet aggregation, we have examined the effect of adenosine receptor function on induced increases of [Ca2+]i by a potent platelet activator, platelet activating factor (PAF). We loaded washed platelets with Fura-2, then induced increases in [Ca2+]i with various concentrations of PAF, and then determined EC50 values (PAF concentration at half-maximal response) and values for maximal response of delta[Ca2+]i (max-delta[Ca2+]i). The EC50 for PAF-delta[Ca2+]i was 112 +/- 37 (SD) pM and the max-delta[Ca2+]i was 284 +/- 138 (SD) nM. Our results show that PAF-delta[Ca2+]i was inhibited in a non-competitive manner by the adenosine receptor agonist cyclohexyladenosine (CHA) with an IC50 of 14.9 microM. This inhibition was partially reversed by theophylline, an adenosine receptor antagonist, with an IC50 of 19 microM. Based on the results of these studies together with evidence from other research groups that platelets do not possess A-1 receptors, our results suggest that CHA inhibited PAF-delta[Ca2+]i in platelets through an activation of A-2 receptors.     

The thrombocyte-activation factor and endotoxin-induced thrombocyte activation

The effect of PAF in aggregation of platelets induced by endotoxin was studied in experiments in vitro. It is indicated that in high concentration (1.10(-7)-1.10(-6) M) PAF did not affect the degree of aggregation of platelets induced by lipopolysaccharides (LPS) S. typhimurium and N. meningitidis. Successive addition to PRP LPS and PAF or joint addition of PAF and LPS did not change the degree of aggregation of each inductor or their sum. A lower concentration of PAF (1.10(-11)-1.10(-9) M) and endotoxin caused a more expressive aggregation of platelets than their successive addition. Stimulating activity of PAF on endotoxin-induced aggregation, perhaps, is caused by involvement of metabolism of arachidonic acid during blood platelets activation.     

Human platelet activation by an alkylacetyl analogue of phosphatidylcholine

The present study has evaluated the influence of semi-synthetic platelet-aggregating factor, (PAF) i.e., alkylacetylglycerophosphocholine, on human platelet morphology, biochemistry and function in order to determine if PAF serves as the corrective factor restoring sensitivity to refractory platelets after treatment with epinephrine. Threshold concentrations of PAF caused irreversible platelet aggregation which could be blocked by agents elevating endogenous levels or cyclic AMP or inhibited by antagonists of platelet prostaglandin synthesis and secretion. PAF did not stimulate platelets through α-adrenergic receptors or receptors for arachidonate, endoperoxides or thromboxanes. 24 h after aspirin ingestion, platelets could be aggregated irreversibly by high concentrations, but not by threshold amounts of PAF, even though they were still insensitive to arachidonate. Another less potent PAF derivative, alkenylacetylglycerophosphocholine, blocked aggregation of 24-h aspirin platelets by PAF, but did not inhibit restoration of arachidonate sensitivity and irreversible aggregation when the samples were treated first with epinephrine. Our findings indicate that threshold amounts of PAF activate human platelets in a physiologic manner and cause irreversible aggregation which is dependent on prostaglandin synthesis and the release reaction. The results do not support the concept that PAF is the mediator of the mechanism of membrane modulation through which epinephrine induces correction of the refractory state in prostaglandin I₂-treated or dissociated platelets, or cells obtained from individuals following aspirin ingestion. Thus, the mechanism of platelet membrane modulation is capable of securing irreversible aggregation of secretion, prostaglandin synthesis or PAF formation.     

Mechanisms of interaction of a plasmalogenic analog of platelet-activating factor with human platelets.

The interaction of a plasmalogenic analog of platelet-activating factor (1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphocholine; 1-alkenyl-PAF) with human platelets was studied. 1-Alkenyl-PAF induced an increase in intracellular Ca2+ concentration and inhibition of adenylate cyclase at significantly higher concentrations than PAF. 1-Alkenyl-PAF inhibits PAF-induced platelet aggregation but has no effect on ADP- or thrombin-induced aggregation of human platelets. In contrast to PAF, 1-alkenyl-PAF increases [3H]PGE1 binding with human platelets. The properties of 1-alkenyl-PAF as an agonist or antagonist of PAF receptors apparently depend on its concentration in the cell medium. Under physiological conditions 1-alkenyl-PAF might be a natural PAF antagonist acting in the human cardiovascular system.     

Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes

A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.     

Alpha-bulnesene, a novel PAF receptor antagonist isolated from Pogostemon cablin

Alpha-bulnesene is a sesquiterpenoid isolated from the water extract of Pogostemon cablin. It showed a potent and concentration-dependent inhibitory effect on platelet-activating factor (PAF) and arachidonic acid (AA) induced rabbit platelet aggregation. In a radioligand binding assay for the PAF receptor, alpha-bulnesene competitively inhibited [(3)H]PAF binding to the PAF receptor with an IC(50) value of 17.62+/-5.68microM. alpha-Bulnesene also dose-dependently inhibited PAF-induced intracellular Ca(2+) increase in fluo-3/AM-loaded platelets (IC(50) values of 19.62+/-1.32microM). Furthermore, alpha-bulnesene inhibited AA-induced thromboxane B(2) (TXB(2)) formation and prostaglandin E(2) (PGE(2)) formation. These results indicate that the inhibitory effect of alpha-bulnesene on platelet aggregation was due to a dual activity; specifically the chemical blocked PAF-induced intracellular signal transduction and interfered with cyclooxygenase activity, which resulted in a decrease in thromboxane formation. This study is the first to demonstrate that alpha-bulnesene is a PAF receptor antagonist as well as an anti-platelet aggregation agent.     

Triazolobenzodiazepines competitively inhibit the binding of platelet activating factor (PAF) to human platelets

PAF causes dose dependent platelet aggregation of human platelet rich plasma or gel filtered platelets (GFP). The benzodiazepines alprazolam and triazolam, but not diazepam (1-10 microM), inhibit PAF induced aggregation but have no effect on aggregation induced by other platelet agonists such as ADP, epinephrine and collagen. The IC50 for aggregation by PAF (4 nM) in GFP is 1 microM for both alprazolam and triazolam. The mechanism for this inhibition was explored by studying the binding of 3H-PAF(0.08 nM) to GFP in Tyrodes buffer containing albumin (0.35%), Mg++ (1mM) and Ca++ (0.5mM). GFP was incubated with different doses of the drug for 5 min prior to addition of 3H-PAF. Incubation was then carried out for 60 min at 25 degrees C to achieve binding equilibrium, as previously established. Alprazolam and triazolam, but not diazepam, caused competitive displacement of 3H-PAF from specific binding sites of GFP. The IC50 of alprazolam was 3.8 microM while that of triazolam was 0.82 microM. Lineweaver-Burk plots of 3H-PAF binding in the presence of inhibitor were also consistent with competitive inhibition. These results are consistent with the interpretation that the specific inhibition of PAF induced platelet aggregation by alprazolam and triazolam, respectively, is due to competitive inhibition of binding of PAF to its receptor.     

A potent inhibitor of platelet activating factor from the saliva of the leech Hirudo medicinalis.

Leech saliva is shown to contain protein platelet aggregation inhibitors and a range of selective low molecular weight (LMW) aggregation inhibitors. Gel filtration on Bio-Gel P-2 (cut-off kDa) yields a protein fraction (Fr. I) and three LMW fractions. Fr. I inhibits aggregation induced by collagen, ADP, epinephrine and arachidonic acid. Of all the fractions, only one, Fr. II (LMW) specifically inhibits aggregation induced by platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine). Fr. II also inhibits thrombin-induced platelet aggregation. Fr. III inhibits aggregation induced by ADP, epinephrine and arachidonic acid, and Fr. IV only that induced by arachidonic acid. Fr. II also inhibits PAF- and thrombin-induced thromboxane generation in platelets, but does not inhibit arachidonic acid-induced thromboxane generation. Efforts to separate the anti-PAF from the anti-thrombin activity have been unsuccessful. The inhibition may therefore be due to a single inhibitor, though it may also be due to several inhibitors. Fr. II also inhibits superoxide anion production in formyl Met-Leu-Phe (fMLP)- and ionophore 23187- stimulated neutrophils. This may be due to the inhibition of the effects of PAF generated within the cell. Preliminary results suggest that the Fr. II inhibitor(s) is (are) amphipathic. The interaction of platelets with PAF and their interaction with the inhibitor(s) are mutually exclusive, and the inhibition may be competitive.     

Platelet aggregation may not be a prerequisite for collagen-stimulated platelet generation of nitric oxide

By determining the sum of the supernatant concentrations of nitrite and nitrate the stimulated generation of nitric oxide (NO) by human washed platelets induced by a range of fibrillar collagen concentrations (0.0156-25 microg ml(-1)) was investigated. Platelet serotonin (5-hydroxytryptamine, 5-HT) efflux and platelet aggregation were also measured. Under resting conditions (0 microg ml(-1) collagen) platelet NO release was equivalent to 1.06+/-0.17 nmol per 10(8) platelets. Maximal NO release, equivalent to 2.1+/-0. 37 nmol per 10(8) platelets, was observed with only 0.0625 microg ml(-1) collagen (P<0.02, stimulated vs. resting release), higher collagen concentrations producing no further increases in platelet NO output. By contrast, maximal platelet aggregation and 5-HT efflux did not occur until collagen concentrations of 2.5 microg ml(-1) and 10-25 microg ml-1), respectively, had been achieved. L-NAME (1 mmol l(-1)) and L-NMMA (1 mmol l(-1)) inhibited stimulated platelet NO generation by 78+/-6% and 72%, respectively. Contrasting with fibrillar collagen, fibrillar beta-amyloid protein had no effect on platelet NO generation, or on 5-HT efflux or aggregation. These data perhaps indicate that NO generation by human platelets is stimulated by concentrations of fibrillar collagen insufficient to elicit an aggregatory response. Such a mechanism could operate in vivo to inhibit platelet aggregation which might otherwise be induced by low concentrations of circulating agonists.     

A specific, photolabile and irreversible antagonist (L662,025) of the PAF-receptor

PAF (0.2 microM) induced maximal platelet aggregation in human PRP and [3H]-PAF (1-5 nM) binding to platelet membrane preparations had Kd value of 3.8 nM and Bmax of 200 fmoles/mg of protein. Without UV irradiation, a synthetic azido tetrahydrofuran derivative L662,025 was a reversible and competitive PAF-receptor antagonist with IC50 values of 5.6 +/- 0.3 microM (platelet aggregation) and 1.0 +/- 0.25 microM (receptor binding). Photolysis of L662,025 in the presence of PRP produced an irreversible inhibition of platelet aggregation and specific binding of [3H]-PAF (1 nM). L662,025 did not affect collagen- or ADP-induced human platelet aggregation before or after photolysis. It is a new probe that can be used to identify and characterize the PAF-receptor.     

Binding of the snake venom-derived proteins applaggin and echistatin to the arginine-glycine-aspartic acid recognition site(s) on platelet glycoprotein IIb.IIIa complex inhibits receptor function

In the present report we describe the platelet-binding characteristics of applaggin and echistatin, potent inhibitors of fibrinogen-dependent platelet aggregation derived from Agkistrodon piscivorus piscivorus and Echis carinatus snake venoms, respectively. Both molecules bound to unstimulated platelets in a specific and saturable manner. At saturation there were 37,100 +/- 3,150 (mean, +/- S.D.) molecules of applaggin and 27,200 +/- 2,816 molecules of echistatin bound/platelet, with dissociation constants (Kd) of 1.4 +/- 0.6 x 10(-7) M and 4.9 +/- 1.2 x 10(-7) M, respectively. Stimulation of platelets with ADP (10 microM) + epinephrine (2 microM) resulted in an increase in the number of molecules bound at saturation to 42,300 +/- 2,105 for applaggin and 32,185 +/- 3,180 for echistatin, with a Kd of 5.6 +/- 0.3 x 10(-8) M and 1.8 +/- 0.6 x 10(-7) M, respectively. The synthetic peptide (Arg)8-Gly-Asp-Val was a competitive antagonist of applaggin and echistatin binding to unstimulated platelets (Ki = 25 and 36 microM, respectively). Applaggin and echistatin inhibited the binding of fibrinogen to stimulated platelets in a dose-dependent manner, with an IC50 of 9 and 25 nM, respectively. In concert with inhibition of platelet aggregation, applaggin and echistatin inhibited platelet secretion and synthesis of thromboxane A2 induced by ADP, collagen, and human gamma-thrombin. The monclonal antibody, LJ-CP3, which inhibits the binding of Arg-Gly-Asp containing ligands to platelet GPIIb.IIIa, also inhibited applaggin binding to unstimulated platelets in a competitive manner (Ki = 4.5 microM). Thus, applaggin and echistatin bind to the platelet GPIIb.IIIa complex, and the Arg-Gly-Asp sequence plays a central role in mediating this interaction.     

Inhibition of Human Platelet Aggregation by Amides and Ester of Salicylic Acid with Platelet-Activating Factor Analogs

The influence of acetyl salicylic acid (ASA) derivatives with platelet-activating factor (PAF) lipid analogs on PAF-induced human platelet aggregation has been studied. It was found that the ASA amide with an ethanolamine plasmalogen PAF analog (1-0-alk-1"-enyl-2-acetyl-sn-glycero-3-phospho-(N-2"-acetoxybenzoyl)ethanolamine) and the ASA ester with a choline plasmalogen PAF analog (1-0-alk-1"-enyl-2-(2"-acetoxybenzoyl)-sn-glycero-3-phosphocholine) at concentrations of 10^–7-10^–6 M effectively inhibit PAF-induced aggregation of human platelets. In contrast to these compounds, the ASA amide with an alkyl PAF analog (1-0-alkyl-2-acetyl-sn-glycero-3-phospho-(N-2"-acetoxybenzoyl)ethanolamine) did not inhibit PAF-induced platelet aggregation. As possible mechanisms of action of the studied compounds, the blockade of PAF-receptor and cyclooxygenase inhibition are proposed.     

Discrepancy of protein kinase C translocation and platelet aggregation--immunocytobiochemical evidence

We searched for possible relationships between platelet aggregation induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) or thrombin and the translocation of protein kinase C. Using monoclonal antibodies against subspecies of protein kinase C, we noted a predominant expression of the isozyme, type II, in human platelets (M. Watanabe, M. Hagiwara, K. Onoda, and H. Hidaka, 1988, Biochem. Biophys. Res. Commun. 152, 642). Analysis of the subcellular distribution of protein kinase C revealed that 65% of the kinase activity was present in the cytosolic fraction in unstimulated platelets, with the remaining activity in the membrane fraction. Treatment of platelets with 100 nM TPA resulted in a greater than 60% decrease in protein kinase C activity in the cytosolic fraction and a greater than 200% increase in the activity in the membrane fraction, within 10 min after treatment. Translocation of the enzyme was also found after treatment of platelets with thrombin, although the response was of lower magnitude than that induced by TPA. Similar results were obtained by immunoblotting using MC-2a, an anti-type II protein kinase C monoclonal antibody. We also examined localization of the enzyme, by electron microscopic immunocytochemistry. The presence of type II protein kinase C seemed to be localized mostly in hyaluromeres and not in granulomeres. When platelets were fixed just after the addition of TPA (within 1 min), protein kinase C was localized at the submembranal region with no remarkable change in shape but there was a decrease in the number of granules in the cytoplasma and the open canalicular system was dilated. We then investigated the effects of cytochalasin B, W-7, ML-9, and H-7 on TPA-induced platelet aggregation and the translocation of protein kinase C. W-7 and ML-9 potently inhibited platelet aggregation but none of these compounds hampered the translocation. Thus, activation of protein kinase C may not be a complete requirement for the initiation of platelet aggregation.     

Nicotinic-type unitary currents in Helix neurons

1. The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10(-4) M), while inducing no aggregation, caused the release of 1248 +/- 1147 pg/ul (mean +/- SD) of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 from stimulated platelet. The addition of 1 x 10(-4) M ADP to platelets caused suboptimal aggregation and the release of only 25 +/- 10 pg TXB2/microliters. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.