Cooperativity between platelet-activating factor and collagen in aggregation of bovine platelets, II

When subthreshold amounts of platelet-activating factor (PAF) and collagen were added simultaneously, strong aggregation of platelets was induced. However, each agonist alone at these concentrations could not induce aggregation at all (S. Kojima et al, (1987) Biochem. Biophys. Res. Commun. 145, 915-920). This cooperativity was observed not only in aggregation but also in changes of intracellular Ca2+ concentration, 47 kDa protein phosphorylation, and formation of thromboxane. These findings suggest that various steps of signal transduction pathways are enhanced during their cooperativity.     

Cooperativity between platelet-activating factor and collagen in platelet aggregation

Cell lysate obtained from cultured vascular endothelial cells contained a substance which induced platelet aggregation. This substance was identified as a phospholipid, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor; PAF), by thin-layer chromatography, phospholipase A2 digestion, inhibition by a specific antagonist, CV-3988, and agonist-specific refractory state. It was further found that PAF and collagen together induced extensive aggregation of platelets even with the concentrations by which each agonist alone could not induce aggregation of platelets at all.     

Release of acetylhydrolase from platelets on aggregation with platelet-activating factor

Acetylhydrolase, the enzyme which inactivates platelet-activating factor (PAF, 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine), was selectively released from bovine platelets by aggregation with physiological concentrations (0.1-10 nM) of PAF with no cell lysis. The release of the acetylhydrolase paralleled that of serotonin. The acetylhydrolase released was active over a broad pH range (pH 5.4-8.6) and was not affected by Ca2+ (1-4 mM) or EDTA (1-8 mM). The Km value of the enzyme was 4.6 microM. Net specific acetylhydrolase activity recovered in the 130,000 x g supernatant after stimulation with PAF could be determined in the presence of EDTA without the activity of Ca2+-dependent phospholipase A2 which was also released from the cells at the same concentration of PAF. The acetylhydrolase was inhibited competitively by specific PAF antagonists, rac-3-(N-n-octadecylcarbamoyloxy)-2-methyoxypropyl-2-thiazolioe thyl phosphate (CV-3988) and (2RS)-1-O-hexadecyl-2-O-ethyl-3-O-(7-thiazolinoheptyl)-glycerol methanesulfonate (ONO-6040). Their Ki values for the enzyme were 1.17 microM and 0.84 microM, respectively. The release of the enzyme could also be detected when the platelets were aggregated with ADP (2.3 microM) or thrombin (0.5 unit). These results suggest that the enzyme released from the aggregated platelets to the blood plasma may also have a physiological function cooperating with the plasma acetylhydrolase.     

Characterization of platelet-activating factor receptors in porcine platelets

Despite a large number of studies describing the properties and effects of platelet-activating factor (PAF), little is known about its receptor structure. The characterization of the PAF receptor from additional cell types and species is important for the design of strategies to purify and characterize the receptor molecule. Porcine platelets were shown to bind PAF with characteristics similar to several other species, based on receptor number, affinity, and the activity of PAF antagonists. We found that the affinity for binding was higher in porcine than in rabbit platelets (Kd = 0.68 +/- 0.13 nM for rabbit and 0.29 +/- 0.10 nM for porcine). Porcine platelets have approximately 281 +/- 158 receptors per cell compared with 689 +/- 229 receptors in rabbit platelets. Rabbit platelets respond to concentrations of PAF that are approximately 10(5)-fold lower than those required for aggregation of porcine platelets, but this difference is probably not due to the differences in receptor number alone. When binding was compared between purified membranes from these two cell types, porcine platelets had 20-fold fewer receptors per milligram of membrane protein, but this difference may have been due to an artifact of the membrane preparation procedure. Binding of PAF was severely hindered at cold temperatures. It was undetectable in whole cells on ice and greatly reduced with purified membranes. This study is the first to characterize PAF receptors in porcine platelets, which represent a potentially useful source of receptor for further biochemical characterization.     

Production of platelet-activating factor by washed rabbit platelets

Production of platelet-activating factor by washed rabbit platelets under stimulation with the ionophore A23187 was investigated utilizing two groups of platelet preparations. The first platelet preparation contained 0.03 +/- 0.02% contaminating white cells, while the second preparation contained 0.48 +/- 0.27% white cells. The latter preparation produced platelet-activating factor, mainly 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 8.3 +/- 6.3 pmol (mean +/- standard deviation) with a range of 2.6 to 21.4 pmol (n = 9), followed by small quantities of 1-octadecenyl- and 1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine. In contrast, there was no production of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by the former platelet preparation having 0.03% leukocytes. These quantitative analyses were carried out by the selected ion monitoring technique and it was concluded that it is necessary to consider the presence of contaminating white cells in studies on the production of platelet-activating factor by platelets.     

Specific binding of phospholipid platelet-activating factor by human platelets

The binding of the phospholipid platelet-activating factor 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC) to washed human platelets was more than 80% complete within 2 min, which coincided with the time of initiation of platelet aggregation by AGEPC. Scatchard plot analysis of the binding of [3H]AGEPC to platelets without and with an excess of unlabeled AGEPC revealed two distinct types of binding sites. One platelet site for AGEPC exhibited a high affinity (KD = 37 +/- 13 nM, mean +/- SD), was saturable, and had a low maximal capacity of 1399 +/- 498 (mean +/- SD) molecules of AGEPC/platelet. The other platelet site demonstrated a nearly infinite binding capacity, consistent with nonreceptor uptake of AGEPC into cellular structures. The specificity of the high-affinity binding site for AGEPC was assessed by comparing the capacity of several analogues of AGEPC to inhibit the binding of [3H]AGEPC to platelets and to induce platelet aggregation. An ether linkage in position 1, a short-chain fatty acid in position 2, and a choline moiety in the polar head group proved to be critical both for the binding of [3H]AGEPC to platelets and for the initiation of platelet aggregation. Exposure of platelets to AGEPC for 5 min at 37 degrees C functionally deactivated the exposed platelets to subsequent stimulation by AGEPC, as assessed by diminished aggregation, and concomitantly reduced the specific binding of [3H]AGEPC. Evaluation of the time course of the events of deactivation revealed the loss of an aggregation response to AGEPC after 90 sec at 37 degrees C, despite the retention of up to 50% of the specific binding sites for AGEPC.     

Stimulation of non-selective cation channels providing Ca2+ influx into platelets by platelet-activating factor and other aggregation inducers.

To elucidate the mechanism of the receptor-stimulated Ca2+ entry into human platelets, the influence of Ca(2+)-mobilizing agonists on plasma membrane potential (Em) has been studied. Em changes were registered using potentiometric probe 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide. The agonist effect on Em varied from hyperpolarization to slight and slow rise. On the contrary, after loading of platelets with intracellular Ca2+ indicator quin2, platelet-activating factor (PAF), thrombin, vasopressin, ADP and thromboxane-A2-mimetic U46619 cause substantial transient membrane depolarization. Similar effects were observed after platelet loading with other Ca2+ chelators fura-2 and indo-1. Agonist-induced depolarization considerably reduced if quin2-loaded platelets were suspended in isoosmotic choline-containing medium. Using Ba2+ as a substitute of Ca2+, we have demonstrated that in choline-containing medium PAF-induced Ba2+ entry into platelets results in membrane depolarization. Dependence on Ba2+ concentration and depolarization kinetics correlates with the dose dependence and kinetics of Ba2+ entry detected by quin2 fluorescence. The agonists also stimulate considerable Na+, Li+ and Cs+ inward currents into platelets. Na(+)-dependent depolarization is 2-5-fold suppressed by extracellular Ca2+ [median inhibitory concentration (IC50) approximately 0.3 mM]. Ni2+ and Cd2+ at similar concentrations block Ca2+ entry and agonist-induced Na2+ current (IC50 for both cations approximately 50 microM). Agonist-induced depolarization is blocked by the adenylate cyclase stimulator prostaglandin E1 and the protein kinase C stimulator phorbol ester. It is concluded that agonists stimulate Ca2+ entry into human platelets via receptor-operated channels which are not strictly selective toward divalent cations and are permeable to Na+, Li+ and Cs+.     

Effect of prostacyclin on platelet-activating factor induced rabbit and platelet aggregation

In this paper, the effect of prostacyclin (PGI2) on the aggregation induced by Platelet-activating factor (PAF), a phospholipid mediator of anaphylaxis, was studied. Synthetic PGI2 and PGI2-like activity generated from rabbit aorta were demonstrated to be effective inhibitors of PAF-induced rabbit platelet aggregation and release of 3H-serotonin (3H-5HT).     

Phosphoinositide breakdown as an indirect link between stimulation and aggregation of rat platelets by thrombin and collagen

When washed rat platelets (1.5 x 10(9)/ml) were stimulated by a threshold concentration of thrombin (0.3 unit/ml) or collagen (10 micrograms/ml), a lag period of about 10 or 30 s, respectively, was seen before the start of aggregation. During the lag period, [32P]phosphatidylinositol 4,5-bisphosphate was degraded as the earliest event within 5-10 s of addition of the stimulus. However, though the extent of phosphatidylinositol 4,5-bisphosphate degradation within 10 s of addition of collagen was greater than that within 20 s of addition of thrombin (0.3 unit/ml), a lag of about 20 s remained before the initiation of aggregation by collagen. This casts doubt on the hypothesis that the stimulus-dependent phosphatidylinositol 4,5-bisphosphate breakdown induces the aggregation of platelets. Phosphatidylinositol labeled with 32Pi or [1-14C]arachidonic acid was scarcely degraded during the lag period. As aggregation proceeded, [14C-arachidonic acid]phosphatidylinositol was degraded with generation of diacylglycerol, phosphatidic acid, arachidonic acid and its metabolites. The maximum aggregation by collagen of rat platelets in which arachidonic acid of phospholipids was replaced in vivo with eicosapentaenoic acid was reduced, but that by thrombin was not, though reduction of thromboxane A2 generation was caused by both stimuli. Indomethacin also fully inhibited the aggregation induced by collagen, but not that induced by thrombin. Hence, thromboxane A2 is required for full aggregation by collagen, but not that by thrombin. These results indicate that thrombin-induced phosphoinositide metabolism may proceed independently of aggregation.     

Effect of prostacyclin on platelet-activating factor induced rabbit platelet aggregation

In this paper, the effect of prostacyclin (PGI₂) on the aggregation induced by Platelet-activating factor (PAF), a phospholipid mediator of anaphylaxis, was studied. Synthetic PGI₂ and PGI₂-like activity generated from rabbit aorta were demonstrated to be effective inhibitors of PAF-induced rabbit platelet aggregation and release of ³H-serotonin (³H-5HT).     

Effects of anions on ADP-stimulated aggregation of bovine blood platelets

ADP-stimulated aggregation of bovine blood platelets was observed in media containing isotonic potassium salts of various monovalent anions. The aggregation depended on the anion in the medium, the order of aggregation being Cl^?, Br^?>I^?>SCN^?, $\text{ClO}{}_4{}^{\mathrm{?}}$. After 30-min incubation, the extent of aggregation of platelets in Cl^? or Br^? medium was little changed, whereas, that in SCN^? or $\text{ClO}{}_4{}^{\mathrm{?}}$ medium was remarkably decreased. This anion dependency of aggregation may be due to change in the membrane potential.     

Radioligand binding of antagonists of platelet-activating factor to intact human platelets

Two new antagonists of platelet-activating factor (PAF), the pyrrolothiazole derivative 52770 RP and the triazolodiazepine WEB 2086, have been studied as radioligands in intact human platelets. [3H]52770 RP and [3H]WEB 2086 bound specifically to high-affinity sites with dissociation constants (Kd) of 14.8 and 6.1 nM, respectively. The maximal number of sites for [3H]52770 RP binding was approx. 15-fold higher than for [3H]PAF and [3H]WEB 2086. In addition, C16-PAF, lyso-PAF, WEB 2086 and 52770 RP had Ki values which were nearly identical for both [3H]PAF and [3H]WEB 2086, whereas only 52770 RP competed for [3H]52770 RP-binding sites. These results demonstrate that in human platelets the sites of [3H]WEB 2086 binding are identical to [3H]PAF-binding sites, whereas those of [3H]52770 RP are not. [3H]WEB 2086 appears, therefore, to be a suitable antagonist radioligand for labelling PAF receptors.     

Platelet aggregation and thromboxane release induced by arachidonic acid, collagen, ADP and platelet-activating factor following low dose acetylsalicylic acid in man

The present study was undertaken in order to characterize the dose-dependent nature of acetylsalicylic acid (ASA) on platelet aggregation and plasma thromboxane B2 (TXB2) release in healthy volunteers. Volunteers received either 25, 50, 100 or 500 mg daily for five consecutive days. At the end of the five day period, all dosages of ASA were capable of completely suppressing TXB2 production and arachidonic acid-induced platelet aggregation. At that time, the second phase of ADP-induced aggregation was also blocked. However, while the inhibition following 500 mg ASA was complete after 24 hours, total inhibition with 100, 50 and 25 mg was attained only after two, three and four days, respectively, indicating the cumulative effect of ASA on platelets. Aggregation induced by collagen was also inhibited dose-dependently- yet slower and at no time complete. ASA had no inhibitory effect on aggregation by platelet-activating factor (PAF). It is concluded that a daily dose of 50 mg ASA would suffice in blocking platelet TXA2 production and aggregation induced by most physiological agents.     

Platelet aggregation and thromboxane release induced by arachidonic acid, collagen, ADP and platelet-activating factor following low dose acetylsalicylic acid in man

The present study was undertaken in order to characterize the dose-dependent nature of acetylsalicylic acid (ASA) on platelet aggregation and plasma thromboxane B₂ (TXB₂) release in healthy volunteers. Volunteers received either 25, 50, 100 or 500 mg daily for five consecutive days. At the end of the five day period, all dosages of ASA were capable of completely suppressing TXB₂ production and arachidonic acid-induced platelet aggregation. At that time, the second phase of ADP-induced aggregation was also blocked. However, while the inhibition following 500 mg ASA was complete after 24 hours, total inhibition with 100, 50 and 25 mg was attained only after two, three and four days, respectively, indicating the cumulative effect of ASA on platelets. Aggregation induced by collagen was also inhibited dose-dependently- yet slower and at no time complete. ASA had no inhibitory effect on aggregation by platelet-activating factor (PAF). It is concluded that a daily dose of 50 mg ASA would suffice in blocking platelet TXA₂ production and aggregation induced by most physiological agents.     

Platelet aggregation induced by platelet-activating factor is suppressed by cystine protease inhibitor

The effect of NCO-700, a cystine protease inhibitor, on platelet-activating factor-induced platelet aggregation was determined. A newly synthesized cystine protease inhibitor (calcium-activated neutral protease and cathepsin B inhibitor), NCO-700 (bis[ethyl (2R, 3R)-3-[(S)-methyl-1-[4-(2,3,4- trimethoxyphenylmethyl) piperazine-1-ylcarbonyl]butylcarbonyl]oxiran-2-carboxy late]sulfate), inhibited platelet-activating factor-induced platelet aggregation. The inhibition was dependent on the preincubation time with NCO-700 and on the concentration of the inhibitor. The release of serotonin was also inhibited almost completely by the 20-min preincubation with 10(-4) M NCO-700. Leupeptin also inhibited platelet-activating factor-induced platelet aggregation. But calcium-activated neutral protease inhibitor did not inhibit it. These observations suggest that NCO-700-sensitive protease(s) such as cystine protease may contribute to platelet aggregation induced by platelet-activating factor.     

Effect of inhaled platelet-activating factor on circulating neutrophils and platelets in vivo and ex vivo in man

We studied the effect of five successive inhalations of platelet-activating factor (PAF) on airway calibre, circulating neutrophil and platelet counts and the activation of these cells ex vivo in normal subjects. PAF (24 ug) caused a mean maximal fall of the expiratory flow rate at 70% vital capacity from a partial manoeuvre (Vp30) of 46.4 +/- 6.2% (p less than 0.001); there was a tachyphylactic response to further doses of PAF. Circulating neutrophil counts fell by 54.3 +/- 10.6% (p less than 0.005) with immediate recovery and with a rebound neutrophilia by the fifth inhalation. Platelet counts showed no significant changes. Aggregation of platelets in platelet-rich plasma to PAF and ADP in vitro at 15 min after the first, second and fifth PAF inhalations was not significantly altered. Chemiluminescence responses of neutrophils to PAF (0.01, 0.1, 1 and 10 microM) in vitro were reduced at 15 min after the fifth PAF inhalation, but this was only significant at 1 microM PAF. Methacholine inhalation did not cause any changes in responsiveness of neutrophils to PAF ex vivo. We conclude that ex vivo platelet desensitisation cannot be used as an index of endogenous PAF release, but reduced responsiveness of neutrophils ex vivo is not a sensitive indicator.     

Effect of inhaled platelet-activating factor on circulating neutrophils and platelets in vivo and ex vivo in man

We studied the effect of five successive inhalations of platelet-activating factor (PAF) on airway calibre, circulating neutrophil and platelet counts and the activation of these cells in normal subjects. PAF (24 ug) caused a mean maximal fall of the expiratory flow rate at 70% vital capacity from a partial manoeuvre (Vp30) of 46.4 ± 6.2% (p < 0.001); there was a tachyphylactic response to further doses of PAF. Circulating neutrophil counts fell by 54.3 ± 10.6% (p < 0.005) with immediate recovery and with a rebound neutrophilia by the fifth inhalation. Platelet counts showed no significant changes. Aggregation of platelets in platelet-rich plasma to PAF and ADP at 15 min after the first, second and fifth PAF inhalations was not significantly altered. Chemiluminescence responses of neutrophils to PAF (0.01, 0.1, 1 and 10 μM) were reduced at 15 min after the fifth PAF inhalation, but this was only significant at 1 μM PAF. Methacholine inhalation did not cause any changes in responsiveness of neutrophils to PAF . We conclude that platelet desensitisation cannot be used as an index of endogenous PAF release, but reduced responsiveness of neutrophils is not a sensitive indicator.     

Binding and internalization of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in washed rabbit platelets

The binding profile of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, platelet-activating factor) to washed rabbit platelets was investigated through the use of structural analogs of AGEPC, e.g. U66985, which specifically suppressed AGEPC biological activities on rabbit platelets. This interaction of AGEPC with platelets could be divided into three different components termed A, B, and C. Component A was considered as one of high affinity (Kd = 0.5 X 10(-9) M) and with a low capacity (about 400 sites/platelet). The binding of AGEPC to component A was reversible and was blocked by the inhibitory analogs of AGEPC. This was considered to be the AGEPC receptor site(s). Component B was irreversible in nature and was presumed to be associated with internalization of AGEPC. The latter process was sensitive to the structural inhibitors. Component C was not affected by the inhibitors and probably represented a nonspecific binding to the lipid layer of the membrane. The binding profile of 1-O-alkyl-2-(lyso)-sn-glycero-3-phosphocholine, a biologically inactive and noninhibitory analog of AGEPC, was observed to consist of a single component and was (also) unaffected by the inhibitors. Internalization of AGEPC into rabbit platelets was further examined by the bovine serum albumin extraction method, which was originally developed by Mohandas et al. (Mohandas, N., Wyatt, J., Mel, S. F., Rossi, M. E., and Shohet, S. B. (1982) J. Biol. Chem. 257, 6537-6543). AGEPC was instantly taken up by the cell and internalization into its membrane, where it remained and was not released into cytosol. The internalization of AGEPC was suppressed by pretreating the cells with AGEPC analogs. In platelets desensitized to AGEPC, no down-regulation of the receptor site(s) was observed. The internalization of AGEPC in the desensitized cells was clearly enhanced and this was obvious even in the presence of the AGEPC inhibitor(s). Even in the presence of the inhibitors, effective internalization of AGEPC was also evident in thrombin-treated cells. These results suggested that the internalization of AGEPC was irreversibly enhanced in the platelets which were activated by AGEPC itself as well as by thrombin.