THROMBOSIS RESEARCH 38; 413-416, 1985 0049-3848/85 $3.00 + .OO Printed in the USA. Copyright (cl 1985 Pergamon Press Ltd. All rights reserved.
RELEASE OF TISSUE-TYPE PLASXINOGEN ACTIVATOR BY PLATELET-ACTIVATING FACTOR
H.-P. Kliicking, F. Markwardt and Anna Hoffmann Institute of Pharmacology and Toxicology, ?Jedical Academy Erfurt, DDR-5010 Erfurt, G.D.R. (Received 2.1.1985; Accepted in original form 7.2.1985 by Editor H. Schroer) I IWRODUCTION
Within the frame;ilorkof our studies on the release of plasminogen activator by mediators (1) we tested the platelet activating factor (PAF, PAF-acether), which is a powerful phospholipid mediator able to provoke platelet (2, 3, 4) and neutrophil activation (5), for its plasminogen activator-releasing effect in the pig ear preparation. To get an idea of the structure-activity relationships and mode of action, analogous compounds and inhibitors of the arachidonate metabolism were used. MATERIALS AND XETHODS The following substances were used: semisynthetic PAFacether: l-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine (consisting of 16.7 % of hexadecyl and 78.2 % of octadecyl chains) (6); rat-PAF: l-O-hexadecyl-2-O-acetyl-rac-glycero-3-phosphocholine (7); rat-iso-PAF: I-0-acetyl-2-0-hexadecyl-rat-glycero-3phosphocholine; rat-lyso-PAF: I-0-hexadecyi-rat-glycero-3-phoshocholine (8); nor-dihydroguiaretic acid (NDGA); mepacrine 7Sigma Chemie GmbH, Xinchen, FRG); acetylsalicylic acid; theophylline; p-aninomethylbenzoic acid (PA&BA) (VEB Pharmazeutisches Kombinat Germed, Dresden, GDR). 1Jodified Tyrode's solution: NaCl 8 g, KC1 0.2 g, NaHCO 1 g, NaH2PO4 0.05 g, glucose 1 g, diluted nith H20 to a vol 2me of 1000 ml, pH adjusted to 7.35 with HCl. Antiserum: IgG antibodies against plasninogen activator produced by an established human melanoma cell line (Dr. P. B. Rifkin, Rockefeller University, New York, NY, USA) prepared by research laboratories of the Department of Obstetrics and Gynecology, University of Lund, Sweden. Activator release VJas studied at constant perfusion volume in the isolated perfused pig ear [for test design see (1, 9):. Key ;,vords:PAF-acether, plasminogen activator release 413
PAF AND PLASMINOGEN ACTIVATOR
Plasminogen activator content was estimated by the fibriyolytic activity appearing during application of the perfusate on plasminogen-containing fibrin-agar plates and converted into Ploug units by means of a urokinase calibration curve (I, 10). Statistical significance was calculated by Student’s t-test. p>O.Oy was considered to be not significant. P&F-acether and the PAF-analogues were dissolved in albuminto a final concensaline (pII 7.4, 2.5 mg human serum albumin/ml) tration of 0.11 mmol/l, stored at -20 OC and further diluted ;;iith albumin-saline immediately before use. RESULTSAND DISCUSSION PAF-acether at concentrations of 10B1l to 5 IO-9 mol/l caused an increase in plasminogen activator release in the isolat d perfused pig ear. At PAP-acether concentrations above 5 IO- % mol/l perfusion :7as decreased due to asoconstriction. RacPAP was effective at concentrations of IO- Es mol/l. Changing of of rat-PAF (rat-iso-PAF) reduced the the Cq and C substituents activator-re it was much lower tvhen the acetyl ? easing activity; group in PAF-acether (rat-lyso-PAF) was removed (Table I). ??
TABLE I Influence of PAP-acether and Its Structural Analogues on Activator Release in the Isolated Perfused Pig Ear Substance
Concentration in perfusion solution (mol/l>
Uumber of experiments (n)
Increase in activator release (%)
Control PAP-ace ther
+, 6 + 62
n. s. S.
The fibrinolytic activity of the venous perfusate is due to since the perfusates did the release of plasminogen activator, not exhibit lytic activity on PAUBA-containing or plasminogenplasfree fibrin plates. Quenchin g with anti-human tissue-type minogen activator showed that the activator released by PAFacether is tissue-type plasminogen activator. Our results obtained in pig ear preparations were in accordance with those obtained by other workers in studies on the release of plasminogen activator by PAP-acether in the perfused rat hind leg and in the ivhole rat (11). Among the substances tested (1, 9, 12, 13) PAP-acether possessed the most pronounced activator-releasing effect. The mech-
PAF AND PLASMINDGEN ACTIVATOR
anism of release has not yet been clarified. In the case of perfusion with T rode's solution, which did not contain Ca++ and Mg++ ions, PA5 -acether did not enhance activator release. The release xas inhibited by the phospholipase inhibitor mepacrine, the lipoxygenase inhibitor NDGA and acetylsalicylic acid. Therefore, the arachidonate metabolism is assumed to be involved in the release reaction. Following additional perfusion with theophylline, which acts by inhibiting phosphodiesterase activity, activator release was inhibited (Table II). TABLE II Influence on the P&F-induced Increase in Plasminogen Activator Release Substance
Concentration in perfusion solution (mol/l)
l$~f$$o~ release (70)
101; + 10
PAF-acether+ PAF-acether + theophylline PAF-acether + mepacrine PAF-acether
+ 10 5
PAF-acether + acetylsalicylic acid
Number of experiments (n)
10:; + 10 ??
322 + 178
+ in modified Tyrode's solution By comparison of the effects of PAF-acether on activator release and platelet aggregation with those of analogous compounds, similar structure-activity relationships were found. In human citrated plasma, the platelet-a gregating effect of PAF did not differ from that of rat-PAF (147 . Compared with PAF, about 10 times higher concentrations of rat-iso-PAF were necessary to produce the same platelet-a gregating effect, whereas rat-lyso-PAF was nearly ineffective (15 f . PA&induced release and aggregation required the presence of Ca++ ions. These reactions were inhibited by theophylline, which elevates the intracellular level of [email protected]
(16). REFEHENCES 1.
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PAF AND PLASMINOGEN ACTIVATOR
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