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p-Aminobenzoic Acid and Its Metabolite p-Acetamidobenzoic Acid Inhibit Agonist-Induced Aggregation and Arachidonic Acid-Induced [Ca2+]i Transients in Human Platelets
Thrombosis Research 95 (1999) 235–243
REGULAR ARTICLE
p-Aminobenzoic Acid and Its Metabolite p-Acetamidobenzoic Acid Inhibit Agonist-Induced Aggregation and Arachidonic Acid-Induced [Ca21]i Transients in Human Platelets Bruno Barbieri1, Rigmor Stain-Malmgren2 and Nikos Papadogiannakis3 Vitamex/Numico, SE-601 16 Norrko¨ping, Sweden; 2Department of Clinical Neuroscience, St: Go¨rans Hospital, Karolinska Institutet, SE-112 81 Stockholm, Sweden; 3Department of Immunology, Microbiology, Pathology and Infectious Diseases, Huddinge University Hospital, Karolinska Institutet, SE-141 86 Huddinge, Sweden. 1
(Received 18 November 1998 by Editor B. Østerud; revised/accepted 8 February 1999)
Abstract We have previously found that the naturally occurring amine p-aminobenzoic acid (PABA) inhibits the thrombin-induced thromboxane B2 production in human platelets. In this report we show that PABA and its acetylated metabolite p-acetamidobenzoic acid (PACBA) inhibit platelet aggregation induced by agonists such as adenosine diphosphate (ADP) and arachidonic acid (AA). Both substances were equipotent to acetylsalicylic acid regarding inhibition of ADP-induced aggregation and approximately 50% as potent as acetylsalicylic acid regarding arachidonic acid-induced aggregation. Although not significantly inhibiting collagen aggregation, PABA and PACBA reduced the concomitant adenosine triphosphate (ATP) secretion by approximately 30 and 20%, respectively. The antiaggregatory effect does not seem to be mediated through cyclic adenosine monophosphate Abbreviations: PABA, p-aminobenzoic acid; PACBA, p-acetamidobenzoic acid; ASA, acetylsalicylic acid; Tx, thromboxane; PRP, platelet-rich plasma; HOST, hypoosmotic shock treatment; PG, prostaglandin; PBS, phosphate-buffered saline; AA, arachidonic acid; [Ca]i, intracellular calcium; T, light transmission; ADP, adenosine diphosphate; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate. Corresponding author: Bruno Barbieri, Vitamex/Numico, Enebygatan 18, SE-601 16 Norrko¨ping, Sweden. Tel: 146 (11) 230000; Fax: 146 (11) 121379; E-mail: ,[email protected]..
(cAMP) increase because in our experiments PABA and PACBA did not significantly affect cAMP levels. However, we have found that PABA and PACBA inhibit the intracellular aequorin indicated Ca21 transient upon arachidonic acid stimulation. Our results describe a hitherto unknown effect of PABA and PACBA on platelet aggregation. 1999 Elsevier Science Ltd. All rights reserved. Key Words: p-Aminobenzoic acid; p-Acetamidobenzoic acid; Calcium; Aggregation; Platelets
H
uman platelets acetylate p-aminobenzoic acid (PABA), a common constituent of cell culture medium, to p-acetamidobenzoic acid (PACBA) [1,2]. The structural similarity between PABA/PACBA and nonsteroidal antiinflammatory drugs (Figure 1), such as acetylsalicylic acid (ASA), and the previously found inhibitory effect of PABA on thromboxane (Tx) B2 production [3], initiated our investigation on possible effects of PABA/PACBA on platelet function. Except for the role of PABA in folate formation, no specific function has been ascribed to PABA. The substance occurs in food where B vitamins are abundant and especially in “green” vegetables and liver. Some decades ago PABA was extensively studied for its biological significance. In some reports PABA showed a chromotrichial effect on
0049-3848/99 $–see front matter 1999 Elsevier Science Ltd. All rights reserved. PII S0049-3848(99)00044-4
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Fig. 1. Molecular structures of p-aminobenzoic acid and p-acetamidobenzoic acid compared to acetylsalicylic acid.
grey hair. Other reports showed that PABA could be useful in the treatment of vitiligo, leukemia, and rheumatic fever [4]. The mechanism behind the effect ascribed to PABA was seldom understood and statements were also sometimes conflicting. When the role of PABA in folate formation was established, and when it was found that only microorganisms synthesized folic acid, the interest for PABA itself diminished. However, today derivatives of PABA are used as local anaesthetics, for UV protection of the skin, and the acetylated form of PABA, p-acetamidobenzoic acid, is one of the components in the antiviral and immunomodulatory drug Inosine Pranobex.
used. Briefly, to make platelet membrane hyperpermeable, washed platelets were suspended in 0.5 mL of a HOST buffer (10 mM EDTA, 3 mM ATP, 3 mM Hepes, and 60 mg aequorin). After 60 seconds isoosmolarity was restored by addition of 44 mL of 2 M KCl in 2 mM Hepes. After a further 3.5 minutes 0.5 mL of a solution consisting of 20 mM MgCl2, 2 mM ATP, 2 mM prostaglandin (PG) E1 was added and the platelets were left at rest for 5 minutes. Thereafter the platelets received 0.9 mL of an albumin containing Ca21-free Hepes-Tyrodes buffer, washed and resuspended in the same buffer. Platelet cAMP was determined in washed platelets obtained using a modified version of a previously described method [6]. Briefly, PRP was chilled to 48C, supplemented with ice cold EDTA to a final concentration of 5 mM, and centrifuged for 6 minutes at 1790 g. The pellet was then resuspended in the same volume of a platelet buffer composed of 0.496 g bovine serum albumin, 0.990 g glucose, and 0.585 g EDTA in 1 L of phosphatebuffered saline (PBS) [3]. Thereafter the platelets were washed according to the method previously described [6]. From the stock solution thus prepared an appropriate volume was taken to obtain 6003106 platelets per test tube. Before starting the experiments the platelets were washed in PBS (centrifugation for 15 minutes at 580 g and 48C) and finally resuspended in PBS.
1. Materials and Methods
1.2. Platelet Aggregation
1.1. Platelet Preparations
Platelet aggregation induced by ADP/fibrinogen (Sigma Chemical Company, St. Louis, MO, USA/ IMCO Corp. Ltd AB, Stockholm, Sweden) and collagen (Collagen Horm, Hormon-Chemie, Munich, Germany) was determined in PRP. Arachidonic acid (AA)-induced platelet aggregation and intracellular calcium transients ([Ca21]i) were monitored in the aequorin-loaded platelets suspended in Ca21-free Tyrode’s buffer. In all experiments we used a Platelet Intracellular Calcium Aggregometer (PICA, Chrono-Log, Haverton, PA, USA). The stirring rate was 1200 rpm and the paper speed was 2 cm/minute. Aliquots of 980 mL PRP or aequorin-loaded platelets were preincubated with 10 mL of the inhibitors (PABA, PACBA, and ASA), appropriate vehicle or plain buffer (controls) for 10 minutes at 378C prior to the addition of 10 mL of the various agonists. PABA, PACBA, and ASA
Citrated blood (0.315% w/v) was obtained from the blood bank at Stockholm So¨der Hospital. Blood was only collected from male healthy volunteers who claimed to have taken no medication during the last 10 days. Platelet-rich plasma (PRP) was obtained by low-speed centrifugation of the blood in a swing-out rotor at 200 g for 15 minutes in room temperature. The platelet count was adjusted to 2503106/mL by dilution with autologous platelet-poor plasma obtained by centrifugation of PRP at 12000 g for 3 minutes. The platelet counts were obtained using an automatic cell counter (Medonic Ca 270, Bergman Bering, Stockholm, Sweden). Washed and aequorin-loaded platelets were prepared according to a previous report [5]. The hypoosmotic shock treatment (HOST) was
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were dissolved in ethanol (95%) and the dilutions were kept on ice during the experiments. Platelet aggregation was determined in percentage change in light transmission (T) and the maximal light transmission was set with platelet-poor plasma when using PRP, or buffer when using aequorinloaded platelets. The aggregation velocity was determined as percentage increase in T/minutes during the first minute after shape change and maximal aggregation as percentage increase in T when aggregation had reached its final plateau. In collageninduced platelet aggregation the concomitant secretion of ATP from dense granules was measured [7]. Quantification of secreted ATP was achieved by addition of a known quantity of ATP to PRP when the platelet secretion had reached its maximum and by determination of the ratios between sample and standard deflections.
four cycles (liquid nitrogen/508C water) and kept at 2208C until assayed the next day. As soon as they thawed, the samples were centrifuged 15 minutes at 3000 g and aliquots diluted 5–25 times and assayed for their cAMP content with a commercial immunoassay (Amersham, Buckinghamshire, England).
1.4. Statistical Analysis
1.3. Measurement of Platelet cAMP
2.1. Platelet Aggregation
Test tubes with 6003106 platelets in 1 mL of PBS were preheated in a shaking water bath. After addition of 1 mL of PBS (1% ethanol), containing 10 mM theophylline (Sigma) and various concentrations of PABA, PACBA, ASA, or PGE1 (Sigma), the samples were incubated for 10 minutes at 378C. Some of these experiments were also performed without theophylline. In another experiment platelets were preincubated with the substances for 10 minutes and then they were stimulated with thrombin (1U/mL) (Sigma) or AA (13 mM) (Sigma) in the presence of 5 mM theophylline for a further 6 minutes. The possible influence of PABA, PACBA, and ASA on cAMP levels were also tested when platelets were given PGE1 and stimulated with various agonists. Thus platelets were preincubated with the drugs to be tested for 10 minutes, then they were given PGE1 (3.5 mM), incubated for further 3 minutes, and thereafter for a final 3 minutes together with various agonists such as 5 mM ADP together with 0.5 mg/mL human fibrinogen or 50 mM AA or 1U/mL thrombin. In order to exclude any possible effect of the vehicle (ethanol), appropriate controls with plain buffer were performed throughout the experiments. In all the experiments the incubations were terminated by immersing the test tubes into liquid nitrogen. The samples were then freeze-thawed for
Figure 2 shows representative tracings, from one of three experiments, of the effect of the inhibitors on ADP-induced platelet aggregation in PRP. 328 mM PABA and PACBA, similar to ASA in the same concentration, had no impact on the initial aggregation rate but caused inhibition of the second wave of aggregation and reversal of aggregation. Mean values of the effect of PABA, PACBA, and ASA (all at 328 mM) on collagen-induced platelet aggregation are shown in Table 1. PABA and PACBA seemed to exert a slight inhibitory effect on the aggregation rate but the reduction was not statistically significant. PABA and PACBA had no inhibitory effect on maximal aggregation. As expected, ASA caused a significant inhibition of collagen-induced platelet aggregation and reduced ATP secretion with 78.064.0% [from 5.9560.89 nmol ATP in control samples to 1.2060.30 nmol ATP (p50.002) in ASA-treated samples]. In spite of having no significant effect on collagen-induced platelet aggregation, PABA and PACBA reduced platelet ATP secretion by 31.369.0% and 19.165.0% respectively; that is, the secretion was reduced to 4.0960.20 nmol ATP (p50.010) in PABA-treated samples and 4.8260.54 nmol in PACBA-treated samples (p50.038). Mean values from the AA-induced aggregation results are shown in Table 1. Preincubation with
The results presented in the histograms and Table 1 are means6SD. Calculation of standard deviations and paired two-tailed Student’s t-test were performed with a computer program for statistical analysis.
2. Results
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Table 1. Effect of PABA, PACBA, and ASA on collagen- and arachidonic acid (AA)induced platelet aggregation Aggregation rate (T%/min)
Control PABA PACBA ASA
Maximal aggregation (T%)
Collagen 5 mg/mL
AA 38.7 mM
Collagen 5 mg/mL
AA 38.7 mM
94.6622.8 79.961.0 81.6611.8 31.9613.4*
47.1612.8 28.1611.6*** 24.167.4*** >0
84.9612.8 86.2612.6 82.1615.8 37.4613.0**
45.3612.1 26.2615.3*** 21.168.5*** >0
* p, 0.05; ** p , 0.01; *** p , 0.001.
ASA (328 mM) completely inhibited platelet aggregation. PABA and PACBA at the same concentration caused a significant reduction of both the aggregation rate and maximum aggregation. Representative tracings from one of three experiments of AA-induced platelet aggregation and intracellular Ca21 release and the impact of PABA, PACBA, and ASA are shown in Figure 3. All the substances showed a sharp and similar reduction of the intracellular Ca21 release. AA caused an intracellular calcium transient that reached a peak that coincided with platelet aggregation velocity maximum; after which it declined. The mean value of this peak [Ca21]i induced by 38.7 mM AA was 3.461.3 mM. Preincubation of platelets with ASA reduced peak [Ca21]i to 1.560.3 mM (p50.006), a reduction of 51.568.8%. The corresponding values for PABA and PACBA were 2.460.7 mM (p50.010) and 3.061.2 mM (p50.070),
Fig. 2. Representative tracings of platelet aggregation in plasma induced by 5 mM ADP. Prior to agonist addition platelets were incubated with the drugs indicated (328 mM) for 10 minutes at 378C.
respectively, and the percentage of inhibition 24.268.2% and 9.065.3%, respectively. PACBA seemed to have a weaker effect in comparison with PABA but this difference was not statistically significant (p50.062).
2.2. Intraplatelet cAMP The cAMP levels in resting platelets after 10 minutes incubation at 378C (2.660.4 pmol/109 platelets, three donors) were not significantly affected by the presence of PABA or PACBA (328 mM, 1 mM, and 5 mM). As expected, the positive control (PGE1) gave a pronounced increase in cAMP production (159.3635.9 pmol). If phosphodiesterase was inhibited with theophylline (Figure 4), a clear accumulation of cAMP was seen. The deviation from the control value when PABA and PACBA were included in the incubation was not significant. Inhibition of cyclooxygenase (PGH synthase) with ASA did apparently not affect the cAMP levels. In the experiments shown in Figure 5 platelets were stimulated with thrombin or AA in the presence of theophylline after preincubation with the indicated drugs. Thrombin per se did not affect cAMP production and neither did the tested drugs in combination with thrombin. On the contrary, AA doubled the cAMP production (p50.004). This increase in cAMP was inhibited by 328 mM ASA (p50.030). PABA and PACBA at the same concentration as ASA did not affect the cAMP levels. If the platelets were preincubated with the various drugs to be tested (PABA, PACBA, and ASA) and thereafter given PGE1 and finally stimulated with ADP, AA, or thrombin, the only significant effect noticed was the cAMP decreasing effect caused by the agonists. The stimulation in cAMP production caused by PGE1 decreased by on aver-
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Fig. 3. Representative tracings of platelet aggregation and aequorinindicated Ca21 signals induced by 38.7 mM AA. Prior to AA addition platelets were incubated with the drugs indicated (328 mM) for 10 minutes at 378C.
age 20, 38, and 40% due to ADP, AA, and thrombin, respectively. Thus, and in contrast to what is seen in Figure 5, AA administration after PGE1 preincubation leads to a decrease in cAMP levels. Neither of the substances tested showed any significant influence on agonist suppression of the PGE1induced cAMP rise.
3. Discussion Aggregation of platelets is a complex physiological event involving several interacting biological pathways. The aggregation can be induced by agonists (such as thrombin, ADP, collagen), TxA2 and endoperoxides, or their precursor AA. Most agonists transmit their signals through receptors coupled to G proteins regulating adenylyl-cyclase activity or phospholipases directly [8], resulting in a sequence of events associated with platelet activation. The hydrolysis of 4,5-bisphosphate by phospholipase C produces at least two second messengers: inositol 1,4,5-triphosphate, which stimulates a cytosolic Ca21 increase, thereby activating phospholipase A2 resulting in AA release and TxA2 production, and 1,2-diacylglycerol, which activates
protein kinase C. These components act synergistically inducing subsequent platelet responses, such as aggregation and secretion. In the present study, we found no marked differences in effect between PABA, PACBA, and ASA on ADP-induced platelet aggregation. The results were surprising in view of our previous finding [3] that PABA, but not PACBA, was an inhibitor of thrombin-induced TxB2 formation (87% inhibition at 328 mM). Thus, PABA like ASA, but not PACBA, could be expected to interfere with the irreversible phase of ADP-induced platelet aggregation in which TxA2 is formed and thus implies the liberation of endogenous AA [9]. Moreover, we have shown [3] that PABA, in contrast to ASA, only marginally inhibited the metabolism of exogenously added AA, whereas PACBA did not interfere with AA metabolism at all. In the present study, we found that PABA and ASA, but also PACBA, significantly reduced AAinduced platelet aggregation. Furthermore, all substances markedly interfered with intracellular calcium recruitment. Free AA mobilises Ca21 because it is immediately metabolised into endoperoxides and TxA2 and calcium is recruited through both extracellular
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Fig. 4. cAMP levels in platelets after 10 minutes of incubation at 378C with the drugs indicated and in the presence of 5 mM theophylline. Each bar represents the mean6SD of the results obtained with platelets from three donors.
Ca21 entry through the plasma membrane and Ca21 release from intracellular stores [10]. The stable TxA2 receptor agonist U46619 has been shown to deplete intracellular Ca21 stores [11,12]. Moreover, the release of Ca21 is prevented by cyclooxygenase inhibitors but the Ca21 entry is not [10]. As our experiments were performed in absence of extracellular calcium ions, the observed [Ca21]i transients were exclusively derived from intracellular stores. The results suggest that the inhibitory effect of PACBA on AA-induced platelet aggregation and [Ca21]i transients are unrelated to the metabolism of AA. While stimulation of platelets with exogenous AA also induces liberation of a con-
siderable amount of endogenous AA [13], the mechanism for the inhibitory effect of PABA likely involves its inhibition of liberation of endogenous AA. The inhibitory effect of ASA on collagen-induced platelet aggregation reflects the moiety of intracellular signal transduction that is routed through metabolism of endogenously liberated AA, in addition to activation of the phosphoinositide cycle. Although in the present study the mean values did not reach statistical significance, both PABA and PACBA exerted weak inhibitory effects on the aggregation rate. More marked was their effect on platelet ATP-secretion, and PABA exerted a
Fig. 5. cAMP levels in platelets after 10 minutes of preincubation at 378C with the drugs indicated and a further 6 minutes with theophylline (5 mM) together with thrombin (1 U/mL) or AA (13 mM). Each bar represents the mean6SD of the results obtained with platelets from four donors.
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stronger inhibiting effect than PACBA. Collageninduced platelet secretion is dependent on intracellular Ca21 mobilization [14] through metabolites both from the AA and phosphoinositol pathways. In view of our results for PABA and PACBA on AA-induced [Ca21]i transients it seems reasonable to assume that their effect on collagen-induced platelet secretion may have resulted from interference with intracellular calcium mobilization. Increased cAMP levels inhibit most platelet responses, including aggregation, ATP release, phosphoinositide breakdown, and rise of [Ca21]i [15]. cAMP can stimulate uptake of calcium into the dense tubular system and extrusion of calcium from the cell [16,17]. In a previous report [18] where the drug PABA-N-D-Mannoside sodium salt [19] was examined for its effect on cAMP, it was also shown that PABA per se in experiments with or without theophylline increased the cAMP levels manifold. However, the concentration of PABA used (80 mM, which actually is more than its solubility) is high compared to the 328 mM that according to our results affects platelet aggregation. In our experiments, with doses up to 5 mM, with or without inhibition of phosphodiesterase PABA or PACBA, did not significantly raise the cAMP content of platelets. This indicates that the antiplatelet effect of these substances is not mediated by elevation of platelet cAMP levels. Even if drugs increasing cAMP levels are potent antiaggregating agents, the reverse relationship is not obvious. As an example, phentolamine and phenothiazine [20] and vitamin K analogues [21] inhibit platelet aggregation without influencing intraplatelet cAMP levels. According to our experiments ASA, PABA, or PACBA did not either show any stimulating effect on the cAMP levels even if PG and Tx production was triggered by thrombin or AA. On the contrary, if platelets were preincubated with ASA and thereafter with AA, cAMP levels decreased, possibly due to inhibition of the small amounts of PGE2 and PGD2 produced that, like PGE1 and PGI2, are known to stimulate cAMP production. PABA and PACBA did not show this cAMP-decreasing effect. AA per se in fact doubled the cAMP production, which is consistent with previous observations [22], whereas thrombin did not affect cAMP levels at all. In the experiments where the platelets were treated with PGE1, the results were quite different. As expected from other reports [23–25], the PGE1-
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related rise in cAMP was decreased by ADP, thrombin, and under these conditions also by AA. Thus, in order to observe any effect of platelet agonists on cAMP levels, it is apparent that adenylyl-cyclase must be stimulated in some way. However, the drugs tested had no significant effect on the cAMP levels in this system either. Regarding the cAMP-decreasing effect of AA, ADP, and thrombin, it has to be mentioned that it is a complex and sometimes conflicting issue. Some have shown that TxA2, PGH2, and the stabile agonist U46619 decreases the PGE1-induced cAMP increase [24,26]. Others [23,27,28] have not found any effect of U46619. Furthermore, agonists such as ADP and thrombin do, per se, regardless of possible effects of induced TxA2 production, inhibit adenylyl-cyclase through their Gi-coupled receptors [8,29]. A general conclusion could likely be that the importance of an endogenous effector eicosanoid (PG or TxA2) depends on if adenylylcyclase is at high turnover or not (i.e., the net cAMP level observed in a cell) seems to be the result of a need to counteract, where the importance of PG or TxA2 as cAMP modulators is dependent on the preceding cAMP status. The inhibitory effect of PABA and PACBA on platelet aggregation appears to involve an interference with the mobilisation and/or utilisation of intracellular Ca21. For PABA, the previously found inhibitory effect on the AA metabolism is likely the underlying cause and may make an additional contribution to the inhibitory effect. PACBA apparently effects the Ca21 levels without any relation to the AA metabolism. Although the precise mechanism of PABA- and PACBA-mediated inhibition of platelet aggregation requires further study, possible synergistic interaction with other antiplatelet agents may prove the substances or derivatives of them useful as antithrombotic agents. In this context it has to be stated that PABA must be regarded as an harmless substance because it has previously been administered to humans in high doses in the treatment of typhus fever [30] and for investigation of its effect on leucocyte count in cases of chronic myeloid leukemia [31]. We thank Agneta So¨derstedt for invaluable laboratory assistance.
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REGULAR ARTICLE
p-Aminobenzoic Acid and Its Metabolite p-Acetamidobenzoic Acid Inhibit Agonist-Induced Aggregation and Arachidonic Acid-Induced [Ca21]i Transients in Human Platelets Bruno Barbieri1, Rigmor Stain-Malmgren2 and Nikos Papadogiannakis3 Vitamex/Numico, SE-601 16 Norrko¨ping, Sweden; 2Department of Clinical Neuroscience, St: Go¨rans Hospital, Karolinska Institutet, SE-112 81 Stockholm, Sweden; 3Department of Immunology, Microbiology, Pathology and Infectious Diseases, Huddinge University Hospital, Karolinska Institutet, SE-141 86 Huddinge, Sweden. 1
(Received 18 November 1998 by Editor B. Østerud; revised/accepted 8 February 1999)
Abstract We have previously found that the naturally occurring amine p-aminobenzoic acid (PABA) inhibits the thrombin-induced thromboxane B2 production in human platelets. In this report we show that PABA and its acetylated metabolite p-acetamidobenzoic acid (PACBA) inhibit platelet aggregation induced by agonists such as adenosine diphosphate (ADP) and arachidonic acid (AA). Both substances were equipotent to acetylsalicylic acid regarding inhibition of ADP-induced aggregation and approximately 50% as potent as acetylsalicylic acid regarding arachidonic acid-induced aggregation. Although not significantly inhibiting collagen aggregation, PABA and PACBA reduced the concomitant adenosine triphosphate (ATP) secretion by approximately 30 and 20%, respectively. The antiaggregatory effect does not seem to be mediated through cyclic adenosine monophosphate Abbreviations: PABA, p-aminobenzoic acid; PACBA, p-acetamidobenzoic acid; ASA, acetylsalicylic acid; Tx, thromboxane; PRP, platelet-rich plasma; HOST, hypoosmotic shock treatment; PG, prostaglandin; PBS, phosphate-buffered saline; AA, arachidonic acid; [Ca]i, intracellular calcium; T, light transmission; ADP, adenosine diphosphate; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate. Corresponding author: Bruno Barbieri, Vitamex/Numico, Enebygatan 18, SE-601 16 Norrko¨ping, Sweden. Tel: 146 (11) 230000; Fax: 146 (11) 121379; E-mail: ,[email protected]..
(cAMP) increase because in our experiments PABA and PACBA did not significantly affect cAMP levels. However, we have found that PABA and PACBA inhibit the intracellular aequorin indicated Ca21 transient upon arachidonic acid stimulation. Our results describe a hitherto unknown effect of PABA and PACBA on platelet aggregation. 1999 Elsevier Science Ltd. All rights reserved. Key Words: p-Aminobenzoic acid; p-Acetamidobenzoic acid; Calcium; Aggregation; Platelets
H
uman platelets acetylate p-aminobenzoic acid (PABA), a common constituent of cell culture medium, to p-acetamidobenzoic acid (PACBA) [1,2]. The structural similarity between PABA/PACBA and nonsteroidal antiinflammatory drugs (Figure 1), such as acetylsalicylic acid (ASA), and the previously found inhibitory effect of PABA on thromboxane (Tx) B2 production [3], initiated our investigation on possible effects of PABA/PACBA on platelet function. Except for the role of PABA in folate formation, no specific function has been ascribed to PABA. The substance occurs in food where B vitamins are abundant and especially in “green” vegetables and liver. Some decades ago PABA was extensively studied for its biological significance. In some reports PABA showed a chromotrichial effect on
0049-3848/99 $–see front matter 1999 Elsevier Science Ltd. All rights reserved. PII S0049-3848(99)00044-4
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Fig. 1. Molecular structures of p-aminobenzoic acid and p-acetamidobenzoic acid compared to acetylsalicylic acid.
grey hair. Other reports showed that PABA could be useful in the treatment of vitiligo, leukemia, and rheumatic fever [4]. The mechanism behind the effect ascribed to PABA was seldom understood and statements were also sometimes conflicting. When the role of PABA in folate formation was established, and when it was found that only microorganisms synthesized folic acid, the interest for PABA itself diminished. However, today derivatives of PABA are used as local anaesthetics, for UV protection of the skin, and the acetylated form of PABA, p-acetamidobenzoic acid, is one of the components in the antiviral and immunomodulatory drug Inosine Pranobex.
used. Briefly, to make platelet membrane hyperpermeable, washed platelets were suspended in 0.5 mL of a HOST buffer (10 mM EDTA, 3 mM ATP, 3 mM Hepes, and 60 mg aequorin). After 60 seconds isoosmolarity was restored by addition of 44 mL of 2 M KCl in 2 mM Hepes. After a further 3.5 minutes 0.5 mL of a solution consisting of 20 mM MgCl2, 2 mM ATP, 2 mM prostaglandin (PG) E1 was added and the platelets were left at rest for 5 minutes. Thereafter the platelets received 0.9 mL of an albumin containing Ca21-free Hepes-Tyrodes buffer, washed and resuspended in the same buffer. Platelet cAMP was determined in washed platelets obtained using a modified version of a previously described method [6]. Briefly, PRP was chilled to 48C, supplemented with ice cold EDTA to a final concentration of 5 mM, and centrifuged for 6 minutes at 1790 g. The pellet was then resuspended in the same volume of a platelet buffer composed of 0.496 g bovine serum albumin, 0.990 g glucose, and 0.585 g EDTA in 1 L of phosphatebuffered saline (PBS) [3]. Thereafter the platelets were washed according to the method previously described [6]. From the stock solution thus prepared an appropriate volume was taken to obtain 6003106 platelets per test tube. Before starting the experiments the platelets were washed in PBS (centrifugation for 15 minutes at 580 g and 48C) and finally resuspended in PBS.
1. Materials and Methods
1.2. Platelet Aggregation
1.1. Platelet Preparations
Platelet aggregation induced by ADP/fibrinogen (Sigma Chemical Company, St. Louis, MO, USA/ IMCO Corp. Ltd AB, Stockholm, Sweden) and collagen (Collagen Horm, Hormon-Chemie, Munich, Germany) was determined in PRP. Arachidonic acid (AA)-induced platelet aggregation and intracellular calcium transients ([Ca21]i) were monitored in the aequorin-loaded platelets suspended in Ca21-free Tyrode’s buffer. In all experiments we used a Platelet Intracellular Calcium Aggregometer (PICA, Chrono-Log, Haverton, PA, USA). The stirring rate was 1200 rpm and the paper speed was 2 cm/minute. Aliquots of 980 mL PRP or aequorin-loaded platelets were preincubated with 10 mL of the inhibitors (PABA, PACBA, and ASA), appropriate vehicle or plain buffer (controls) for 10 minutes at 378C prior to the addition of 10 mL of the various agonists. PABA, PACBA, and ASA
Citrated blood (0.315% w/v) was obtained from the blood bank at Stockholm So¨der Hospital. Blood was only collected from male healthy volunteers who claimed to have taken no medication during the last 10 days. Platelet-rich plasma (PRP) was obtained by low-speed centrifugation of the blood in a swing-out rotor at 200 g for 15 minutes in room temperature. The platelet count was adjusted to 2503106/mL by dilution with autologous platelet-poor plasma obtained by centrifugation of PRP at 12000 g for 3 minutes. The platelet counts were obtained using an automatic cell counter (Medonic Ca 270, Bergman Bering, Stockholm, Sweden). Washed and aequorin-loaded platelets were prepared according to a previous report [5]. The hypoosmotic shock treatment (HOST) was
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were dissolved in ethanol (95%) and the dilutions were kept on ice during the experiments. Platelet aggregation was determined in percentage change in light transmission (T) and the maximal light transmission was set with platelet-poor plasma when using PRP, or buffer when using aequorinloaded platelets. The aggregation velocity was determined as percentage increase in T/minutes during the first minute after shape change and maximal aggregation as percentage increase in T when aggregation had reached its final plateau. In collageninduced platelet aggregation the concomitant secretion of ATP from dense granules was measured [7]. Quantification of secreted ATP was achieved by addition of a known quantity of ATP to PRP when the platelet secretion had reached its maximum and by determination of the ratios between sample and standard deflections.
four cycles (liquid nitrogen/508C water) and kept at 2208C until assayed the next day. As soon as they thawed, the samples were centrifuged 15 minutes at 3000 g and aliquots diluted 5–25 times and assayed for their cAMP content with a commercial immunoassay (Amersham, Buckinghamshire, England).
1.4. Statistical Analysis
1.3. Measurement of Platelet cAMP
2.1. Platelet Aggregation
Test tubes with 6003106 platelets in 1 mL of PBS were preheated in a shaking water bath. After addition of 1 mL of PBS (1% ethanol), containing 10 mM theophylline (Sigma) and various concentrations of PABA, PACBA, ASA, or PGE1 (Sigma), the samples were incubated for 10 minutes at 378C. Some of these experiments were also performed without theophylline. In another experiment platelets were preincubated with the substances for 10 minutes and then they were stimulated with thrombin (1U/mL) (Sigma) or AA (13 mM) (Sigma) in the presence of 5 mM theophylline for a further 6 minutes. The possible influence of PABA, PACBA, and ASA on cAMP levels were also tested when platelets were given PGE1 and stimulated with various agonists. Thus platelets were preincubated with the drugs to be tested for 10 minutes, then they were given PGE1 (3.5 mM), incubated for further 3 minutes, and thereafter for a final 3 minutes together with various agonists such as 5 mM ADP together with 0.5 mg/mL human fibrinogen or 50 mM AA or 1U/mL thrombin. In order to exclude any possible effect of the vehicle (ethanol), appropriate controls with plain buffer were performed throughout the experiments. In all the experiments the incubations were terminated by immersing the test tubes into liquid nitrogen. The samples were then freeze-thawed for
Figure 2 shows representative tracings, from one of three experiments, of the effect of the inhibitors on ADP-induced platelet aggregation in PRP. 328 mM PABA and PACBA, similar to ASA in the same concentration, had no impact on the initial aggregation rate but caused inhibition of the second wave of aggregation and reversal of aggregation. Mean values of the effect of PABA, PACBA, and ASA (all at 328 mM) on collagen-induced platelet aggregation are shown in Table 1. PABA and PACBA seemed to exert a slight inhibitory effect on the aggregation rate but the reduction was not statistically significant. PABA and PACBA had no inhibitory effect on maximal aggregation. As expected, ASA caused a significant inhibition of collagen-induced platelet aggregation and reduced ATP secretion with 78.064.0% [from 5.9560.89 nmol ATP in control samples to 1.2060.30 nmol ATP (p50.002) in ASA-treated samples]. In spite of having no significant effect on collagen-induced platelet aggregation, PABA and PACBA reduced platelet ATP secretion by 31.369.0% and 19.165.0% respectively; that is, the secretion was reduced to 4.0960.20 nmol ATP (p50.010) in PABA-treated samples and 4.8260.54 nmol in PACBA-treated samples (p50.038). Mean values from the AA-induced aggregation results are shown in Table 1. Preincubation with
The results presented in the histograms and Table 1 are means6SD. Calculation of standard deviations and paired two-tailed Student’s t-test were performed with a computer program for statistical analysis.
2. Results
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Table 1. Effect of PABA, PACBA, and ASA on collagen- and arachidonic acid (AA)induced platelet aggregation Aggregation rate (T%/min)
Control PABA PACBA ASA
Maximal aggregation (T%)
Collagen 5 mg/mL
AA 38.7 mM
Collagen 5 mg/mL
AA 38.7 mM
94.6622.8 79.961.0 81.6611.8 31.9613.4*
47.1612.8 28.1611.6*** 24.167.4*** >0
84.9612.8 86.2612.6 82.1615.8 37.4613.0**
45.3612.1 26.2615.3*** 21.168.5*** >0
* p, 0.05; ** p , 0.01; *** p , 0.001.
ASA (328 mM) completely inhibited platelet aggregation. PABA and PACBA at the same concentration caused a significant reduction of both the aggregation rate and maximum aggregation. Representative tracings from one of three experiments of AA-induced platelet aggregation and intracellular Ca21 release and the impact of PABA, PACBA, and ASA are shown in Figure 3. All the substances showed a sharp and similar reduction of the intracellular Ca21 release. AA caused an intracellular calcium transient that reached a peak that coincided with platelet aggregation velocity maximum; after which it declined. The mean value of this peak [Ca21]i induced by 38.7 mM AA was 3.461.3 mM. Preincubation of platelets with ASA reduced peak [Ca21]i to 1.560.3 mM (p50.006), a reduction of 51.568.8%. The corresponding values for PABA and PACBA were 2.460.7 mM (p50.010) and 3.061.2 mM (p50.070),
Fig. 2. Representative tracings of platelet aggregation in plasma induced by 5 mM ADP. Prior to agonist addition platelets were incubated with the drugs indicated (328 mM) for 10 minutes at 378C.
respectively, and the percentage of inhibition 24.268.2% and 9.065.3%, respectively. PACBA seemed to have a weaker effect in comparison with PABA but this difference was not statistically significant (p50.062).
2.2. Intraplatelet cAMP The cAMP levels in resting platelets after 10 minutes incubation at 378C (2.660.4 pmol/109 platelets, three donors) were not significantly affected by the presence of PABA or PACBA (328 mM, 1 mM, and 5 mM). As expected, the positive control (PGE1) gave a pronounced increase in cAMP production (159.3635.9 pmol). If phosphodiesterase was inhibited with theophylline (Figure 4), a clear accumulation of cAMP was seen. The deviation from the control value when PABA and PACBA were included in the incubation was not significant. Inhibition of cyclooxygenase (PGH synthase) with ASA did apparently not affect the cAMP levels. In the experiments shown in Figure 5 platelets were stimulated with thrombin or AA in the presence of theophylline after preincubation with the indicated drugs. Thrombin per se did not affect cAMP production and neither did the tested drugs in combination with thrombin. On the contrary, AA doubled the cAMP production (p50.004). This increase in cAMP was inhibited by 328 mM ASA (p50.030). PABA and PACBA at the same concentration as ASA did not affect the cAMP levels. If the platelets were preincubated with the various drugs to be tested (PABA, PACBA, and ASA) and thereafter given PGE1 and finally stimulated with ADP, AA, or thrombin, the only significant effect noticed was the cAMP decreasing effect caused by the agonists. The stimulation in cAMP production caused by PGE1 decreased by on aver-
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Fig. 3. Representative tracings of platelet aggregation and aequorinindicated Ca21 signals induced by 38.7 mM AA. Prior to AA addition platelets were incubated with the drugs indicated (328 mM) for 10 minutes at 378C.
age 20, 38, and 40% due to ADP, AA, and thrombin, respectively. Thus, and in contrast to what is seen in Figure 5, AA administration after PGE1 preincubation leads to a decrease in cAMP levels. Neither of the substances tested showed any significant influence on agonist suppression of the PGE1induced cAMP rise.
3. Discussion Aggregation of platelets is a complex physiological event involving several interacting biological pathways. The aggregation can be induced by agonists (such as thrombin, ADP, collagen), TxA2 and endoperoxides, or their precursor AA. Most agonists transmit their signals through receptors coupled to G proteins regulating adenylyl-cyclase activity or phospholipases directly [8], resulting in a sequence of events associated with platelet activation. The hydrolysis of 4,5-bisphosphate by phospholipase C produces at least two second messengers: inositol 1,4,5-triphosphate, which stimulates a cytosolic Ca21 increase, thereby activating phospholipase A2 resulting in AA release and TxA2 production, and 1,2-diacylglycerol, which activates
protein kinase C. These components act synergistically inducing subsequent platelet responses, such as aggregation and secretion. In the present study, we found no marked differences in effect between PABA, PACBA, and ASA on ADP-induced platelet aggregation. The results were surprising in view of our previous finding [3] that PABA, but not PACBA, was an inhibitor of thrombin-induced TxB2 formation (87% inhibition at 328 mM). Thus, PABA like ASA, but not PACBA, could be expected to interfere with the irreversible phase of ADP-induced platelet aggregation in which TxA2 is formed and thus implies the liberation of endogenous AA [9]. Moreover, we have shown [3] that PABA, in contrast to ASA, only marginally inhibited the metabolism of exogenously added AA, whereas PACBA did not interfere with AA metabolism at all. In the present study, we found that PABA and ASA, but also PACBA, significantly reduced AAinduced platelet aggregation. Furthermore, all substances markedly interfered with intracellular calcium recruitment. Free AA mobilises Ca21 because it is immediately metabolised into endoperoxides and TxA2 and calcium is recruited through both extracellular
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Fig. 4. cAMP levels in platelets after 10 minutes of incubation at 378C with the drugs indicated and in the presence of 5 mM theophylline. Each bar represents the mean6SD of the results obtained with platelets from three donors.
Ca21 entry through the plasma membrane and Ca21 release from intracellular stores [10]. The stable TxA2 receptor agonist U46619 has been shown to deplete intracellular Ca21 stores [11,12]. Moreover, the release of Ca21 is prevented by cyclooxygenase inhibitors but the Ca21 entry is not [10]. As our experiments were performed in absence of extracellular calcium ions, the observed [Ca21]i transients were exclusively derived from intracellular stores. The results suggest that the inhibitory effect of PACBA on AA-induced platelet aggregation and [Ca21]i transients are unrelated to the metabolism of AA. While stimulation of platelets with exogenous AA also induces liberation of a con-
siderable amount of endogenous AA [13], the mechanism for the inhibitory effect of PABA likely involves its inhibition of liberation of endogenous AA. The inhibitory effect of ASA on collagen-induced platelet aggregation reflects the moiety of intracellular signal transduction that is routed through metabolism of endogenously liberated AA, in addition to activation of the phosphoinositide cycle. Although in the present study the mean values did not reach statistical significance, both PABA and PACBA exerted weak inhibitory effects on the aggregation rate. More marked was their effect on platelet ATP-secretion, and PABA exerted a
Fig. 5. cAMP levels in platelets after 10 minutes of preincubation at 378C with the drugs indicated and a further 6 minutes with theophylline (5 mM) together with thrombin (1 U/mL) or AA (13 mM). Each bar represents the mean6SD of the results obtained with platelets from four donors.
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stronger inhibiting effect than PACBA. Collageninduced platelet secretion is dependent on intracellular Ca21 mobilization [14] through metabolites both from the AA and phosphoinositol pathways. In view of our results for PABA and PACBA on AA-induced [Ca21]i transients it seems reasonable to assume that their effect on collagen-induced platelet secretion may have resulted from interference with intracellular calcium mobilization. Increased cAMP levels inhibit most platelet responses, including aggregation, ATP release, phosphoinositide breakdown, and rise of [Ca21]i [15]. cAMP can stimulate uptake of calcium into the dense tubular system and extrusion of calcium from the cell [16,17]. In a previous report [18] where the drug PABA-N-D-Mannoside sodium salt [19] was examined for its effect on cAMP, it was also shown that PABA per se in experiments with or without theophylline increased the cAMP levels manifold. However, the concentration of PABA used (80 mM, which actually is more than its solubility) is high compared to the 328 mM that according to our results affects platelet aggregation. In our experiments, with doses up to 5 mM, with or without inhibition of phosphodiesterase PABA or PACBA, did not significantly raise the cAMP content of platelets. This indicates that the antiplatelet effect of these substances is not mediated by elevation of platelet cAMP levels. Even if drugs increasing cAMP levels are potent antiaggregating agents, the reverse relationship is not obvious. As an example, phentolamine and phenothiazine [20] and vitamin K analogues [21] inhibit platelet aggregation without influencing intraplatelet cAMP levels. According to our experiments ASA, PABA, or PACBA did not either show any stimulating effect on the cAMP levels even if PG and Tx production was triggered by thrombin or AA. On the contrary, if platelets were preincubated with ASA and thereafter with AA, cAMP levels decreased, possibly due to inhibition of the small amounts of PGE2 and PGD2 produced that, like PGE1 and PGI2, are known to stimulate cAMP production. PABA and PACBA did not show this cAMP-decreasing effect. AA per se in fact doubled the cAMP production, which is consistent with previous observations [22], whereas thrombin did not affect cAMP levels at all. In the experiments where the platelets were treated with PGE1, the results were quite different. As expected from other reports [23–25], the PGE1-
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related rise in cAMP was decreased by ADP, thrombin, and under these conditions also by AA. Thus, in order to observe any effect of platelet agonists on cAMP levels, it is apparent that adenylyl-cyclase must be stimulated in some way. However, the drugs tested had no significant effect on the cAMP levels in this system either. Regarding the cAMP-decreasing effect of AA, ADP, and thrombin, it has to be mentioned that it is a complex and sometimes conflicting issue. Some have shown that TxA2, PGH2, and the stabile agonist U46619 decreases the PGE1-induced cAMP increase [24,26]. Others [23,27,28] have not found any effect of U46619. Furthermore, agonists such as ADP and thrombin do, per se, regardless of possible effects of induced TxA2 production, inhibit adenylyl-cyclase through their Gi-coupled receptors [8,29]. A general conclusion could likely be that the importance of an endogenous effector eicosanoid (PG or TxA2) depends on if adenylylcyclase is at high turnover or not (i.e., the net cAMP level observed in a cell) seems to be the result of a need to counteract, where the importance of PG or TxA2 as cAMP modulators is dependent on the preceding cAMP status. The inhibitory effect of PABA and PACBA on platelet aggregation appears to involve an interference with the mobilisation and/or utilisation of intracellular Ca21. For PABA, the previously found inhibitory effect on the AA metabolism is likely the underlying cause and may make an additional contribution to the inhibitory effect. PACBA apparently effects the Ca21 levels without any relation to the AA metabolism. Although the precise mechanism of PABA- and PACBA-mediated inhibition of platelet aggregation requires further study, possible synergistic interaction with other antiplatelet agents may prove the substances or derivatives of them useful as antithrombotic agents. In this context it has to be stated that PABA must be regarded as an harmless substance because it has previously been administered to humans in high doses in the treatment of typhus fever [30] and for investigation of its effect on leucocyte count in cases of chronic myeloid leukemia [31]. We thank Agneta So¨derstedt for invaluable laboratory assistance.
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