Lipoxygenase products: Leukotrienes C4, D4, A4's breakdown products and 12-HPETE influence platelet aggregation in vivo

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LIPOXYGENASE PRODUCTS: LEUKOTRIENES C4, D4, A4's BREAKDOWN PRODUCTS AND 12-HPETE INFLUENCE PLATELET AGGREGATION IN VIVO. K.S.Herrmann* Institute of Pharmacology,

Bayer AG, D-56 Wuppertal,

*present address: Dept. of Cardiology, GEttingen, D-34 Gijttingen, FRG

University

FRG of

ABSTRACT Clinical and oatholoaical observations of diseases concomitant which leukotriene release show consistently an This effect was involvement of platelet activation. hitherto believed to be due to tissue trauma. The aim of the present study was to elucidate whether lipoxygenase products such as leukotrienes C4, D4 , 12-HPETE and the breakdown products of leukotriene A4 have a direct proPlatelet aggregation aggregatory property of their own. was induced by intravascular excitation of fluoresceinisothiocyanate-dextran in arterioles of the hamster cheek "Time to aggregate appearance" was assessed prior pouch. and after parenteral application of the studied compounds. LTA4's breakdown products were found to have antiaggregatory properties, whereas LTC4, D4 and 12-HPETE enhanced platelet aggregability. INTRODUCTION Leukotrienes (LTs) form a recently discovered group of hydroperoxy derivatives which are produced from arachidonic acid, an ubiquitous part of cell membranes. Arachidonic acid is released upon attack by a phospholipase and is further enzymatically metabolized to the different prostaglandins or, by lipoxygenases, to the LTs. 5-hydroperoxyeicosatetraenoic-acid is formed, which then is converted to the epoxide leukotriene A4 (LTA4). LTA4 may add to form leukotriene B4 or add glutawater enzymatically thione to form leukotriene C4 (LTC4) which in turn may be Various cells have converted to leukotriene D4 (LTD4). been demonstrated to produce LTs (l-5) among which the leukocytes attracted the attention of most laboratories. LTs have been shown to comprise the major biological "slow-reacting substance of activity of the so-called anaphylaxis" (SRS-A), which belongs to a group of compounds which are generated immunologically after antigen challenge (3,4). LTs respective spectra of biological properties have been so far described to mainly compromise: 1) for LTB4 : chemokinetic and aggregating properties for chemotactic, leukocytes, enhancement of plasma exudation; 2) for LTC4

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as well as LTD4: contraction of smooth muscle of blood small airways, inducing leakage from venules vessels and (6 and 7 give excellent reviews). Some clinical observations on antigen-induced asthma and acute myocardial infarction suggest, that leukotrienes may have also effects on platelet functions in vivo. Both diseases are thought to be accompanied byLTylease, (6,7) and both of them are found to be accompanied by platelet activation (8-11). As no direct in vitro effect of leukotrienes on platelets has been demonstrated yet, platelet involvement has been thought to be secondary to the concomitant tissue trauma and endothelial cell alteration. What kinds of leukotrienes are formed from ,arachidonic acid does not only depend on the cell type, but also varies with incubation conditions (12,13). To circumvent these difficulties, we tested the impact of these compounds on platelet aggregation in vivo. Vessels of the -hamster cheek pouch were prepared for intravital microscopy and platelet aggregation was induced by intravascular excitation of fluoresceinisothiocyanate-dextran ( FITC-d ) (14). This method initiates aggregation by selectively damaging endothelial cells in the area of FITC-d excitation (15). We measured the time elapsing from onset and of the noxious stimulus until the first aggregate was seen to adhere to the vascular endothelium (TAA=TAA was measured before time to aggregate appearance). and after parenteral application of the respective compound, which was handed blind to the assistant. METHODS Syrian hamsters of one breed, weighing 80-100 g were used. Pentobarbital, 60 mg/kg bw. was given intraperitoneally and supplemented when required. A small canula was inserted into one of the subclavian arteries for injections of the FITC-d or drugs. The animal was placed on a microscopic stage. The right cheek pouch was gently everted and pinned into a circular well, the central part of which served as a viewing pedestal for the pouch. To keep the tissue moist, the pouch was continuously flushed with a solution at a constant rate of 5 ml/min. This superfusate was freshly prepared each day from concentrated stock solutions to give the following concentrations: NaCl 131.9 mM , KC1 4.7 mM , CaCl x 2H20 2 mM , MgS04 x 7H20 1.2 mM NaHC03 18 mM. The superfusate was continuously bubbled kith 95% N2 and 5% CO2 to give a pH of 7.35. The temperature of the liquid in the well was maintained at 370C. A small crescent-shaped flap was cut into the upper layer of the cheek pouch and, using watchmakers forceps and iridectomy scissors, a single-layer preparation was fashioned. tissue under a About 1 cm2 was cleared of connective

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stereomicroscope at 25x magnification within 15-20 min, then the animal was transferred to the microscope. A small thermistor was put into the left cheek pouch to measure body temperature and drive a closed circuitcontrolled IR lamp. An intravital microscope (Orthoplan, Leitz, Wetzlar, FRG) permitting trans- as well as epiillumination was used. A condenser with long working distance and a numerical aperture of 0.6 was used to transilluminate the object with a stabilized halogen lamp of 100 W. The Ploemopak system which utilizes the objective for both epi-illumination and observation, was used to induce fluorescence with a stabilized high-pressure mercury lamp HBO-100. An appropriate set of filters (K2, Leitz,Wetzlar, FRG) separates light at different wavelengths for excitation and observation. For general observation of the microvessels, we employed 10 x and 20 x objectives whereas a UO 75W objective with a numerical aperture of 0.9 was used for measurements. We used pentobarbital (Nembutal,60 mg/ml,Abbott,USA) for anesthesia. FITC-dextran (Sigma,USA) with an average molecular weight of 64,400 and a degree of substitution of 5 FITC molecules/1000 glucose molecules was used. Maximum absorption was at 490 nm, maximum emission at 510 nm in vitro. Stock solutions of LTC4, LTD4, and 12-HPETE were prepared in ethanol and kept at -8O'C. LTA4 lithium salt was similarly stored, but being unstable, a mixture of its spontanuous breakdown products 5,6 di-HETE and isomers I and II of LTB4 were thus finally tested. As controls we tested carrier alone and, as a vasoconstrictor, a bolus of 10 microgram/kg angiotensinamid (Hypertensin CIBA). These solutions were given blind to the assistant who evaporated the ethanol and prepared the required dilutions in Tyrode immediately before application. The compounds were administered as bolus injections. The mean and standard error of the mean were calculated, the significance of difference was calculated with the U-test of Wilcoxon, Mann and Whitney. Experimental Protocol: 30 min after surgery, 0.3 ml 'of a 5% FITC-d solution was injected into the subclavian artery. Only preparations with brisk flow in all vessels and without obvious leakage were used. In transil.lumination, an arteriole with a diameter between 11 and 15 pm was centered in the field of observation and then FITC was excited by switching over to epiillumination. Using stop watches, the time elapsing between onset of the.noxious stimulus and appearance of the first platelet aggregate adhering to the vessel wall was determined (TAA=time to aggregate appearance). TAA was assessed before application and within the following 5-15 min after application of the respective compound when vasoconstriction had ceased and

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normal blood flow was regained. Flow was originally esti"4" represented mated using a four step gradation where the normal, brisk flow. Later, this grade 4 velocity was measured to represent at least 1.8 mm/set of centerline velocity (16). Six animals were used in each group. RESULTS Both controls, carrier alone and a bolus of 10 ug/kg angiotensinamid as a vasoconstrictor did not influence The time to aggregate appearance (TAA) significantly. results obtained with the tested compounds are depicted in fig 1. : After application of LTA4's break-down products, the mean TAA did not differ significantly from the control after 10-8 and 10-7 g/kg bw of originally prepared LTA4. Being preteated with 10e5 g/kg bw however, TAA was significantly delayed (~~0.002). TAA was After 1O-7 s/kg bw LTC4, D4 or 12-HPETE, significantly accelerated (p
I

'LTA:

150_

I

loo-

T PfJ

fi f

50-

01 Fig.

I

0

control

10-1

10-6

*

10" [g/kg1

>Platelet aggregation assessed in the hamster cheek pouch by intravascular excitation of the fluorescent macromolecule fluoresceinisothiocyanate-dextran (FITC-d) before and after parenteral bolus injection of spontanous of leukotrienes C4, D4 , a mixture breakdown products of LTA4 (nLTA411) and 12-HPETE. Means and SEM are shown.

DISCUSSION an immediate and dose deOthers have demonstrated pendent vasoconstriction of hamster cheek pouch vessels

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after addition of LTC4 or LTD4 to the superfusate (17). vasoconstriction was also observed during our experiments after parenteral application, but our interest was not focussed on vasoconstriction and hence was not quantified. vasoconstriction however limited the range of dosages to be studied, as after boli with dosages higher than 10m5 g/kg b.w., hamsters did not regain normal blood flow which is a prerequisite for reliable measurements with the model used. 12-HPETE was found to have a clear dose dependent This compound is generated by proaggregatory property. is liberated during platelet lipoxygenase (18) and 12-HPETE was proposed to platelet aggregation (19). inhibit platelet cycle-oxygenase and was shown to inhibit platelet aggregation in vitro (20,21). As platelet cyclooxygenase synthesizes>h~aggregatory thromboxane, its inhibition should indeed result in an antiaggregatory Later it was shown that this compound blocks effect. vascular synthesis of the potent antiaggregatory prostacyclin (22), and this effect may be of major importance in vivo, as shown by our results. For LTA4's breakdown products, no significant influence on aggregation could be found for doss es up to 1O-6 g/kg bw. of the original LTA4. With IO- ? g/kg bw however, TAA was prolonged to 176.6% of the control value. that at least one of its breakdown This indicates, products exerts antiaggregatory properties. LTC4 and LTD4, both belonging to the cysteinyl leukotrienes, were found to exert a clear-cut dose dependent proaggregatory effectivity in the dosages tested. Their dose-response curves are nearly identical, with 10m5 g/kg bw, aggregation is twice as fast as in controls. The presented results demostrate that leukotrienes may have proaggregatory as well as antiaggregatory effects on platelet functions in vivo. As this may be of importance in pathological situ~~~~ns, the involved mechanism and its influencibilty deserves further investigations.

1.

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Austen,K.F.,

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Lewis,R.A., Drazen,J.M.,Austen,K.F., Clark,D.A. and Corey,E.J.: Identification of the C(6)-S-conjugate of leukotriene A with cysteine as a naturally occurring slow reacting substance of anaphylaxis (SRS-A). Biochem. Biophys. Res. Comm. 96: 271-277 (1980)

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Knauer,K.A., Lichtenstein,L.M., Adkinson,F.N.Jr., Fish,J.E.: Platelet activation during antigen-induced airway reactions in asthmatic subjects. N. Eng1.J. Med. 304: 1404-1407 (1981)

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Jazayeri,M.R., Reen,B.M., Edwards,J.A.: Asthma induced myocardial infarction in a patient with normal coronary arteries: a case report and a pathogenetic hypothesis. J. Med. 14: 351-361 (1983)

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Sorkin,R.P., Tokarsky,J.M., Huber-Smith,M.J., Steiger,J.F., McCann,D.S.: In vivo platelet aggregation and plasma catecholamines in acute myocardial infarction. Am. Heart J. 104: 1255-1261 (1982)

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Morris,H.R., Taylor,G.W., Piper,P.J., Samhoun, M.N. and Tippins,J.R.: Slow reacting substances (SRSs), the structure identification of SRSs from rat basophil leukaemia (RBL-1) cells. Prostaglandins 19: 185-201 (1980)

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Ford-Hutchinson,A. W., Piper,P.J. and Samhoun,M.N.: Generation of leukotriene B4 all trans isomers and 5-hydroxyeicosatetraenoic acid by rat basophilic

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Herrmann,K.S.: Platelet aggregation induced in the hamster cheek pouch by a photochemical process with excited fluoresceinisothiocyanate dextran. Microvasc. Res. 26: 238-249 (1983)

15.

Herrmann,K.S. and Voigt,W.-H.: Ultrastructural observations of an electron dense amorphous layer on selectively damaged endothelial cells, a possible trigger of in vivo, and its inhibithrombogenesis tion by nafazatrom.Thrombosis Res. 36: 205-215 (1984)

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Dahlen,S.-D., Bj6rg,J., Hedqvist,P., Arfors,K.-E., Hammarstriim,S., and Lindgren,J.A. Samuelsson,B.: Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules. Proc. Natl. Acad. Sci. USA 78: 3887-3891 (1981)

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Hamberg,M. and Samuelsson,B.: Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Natl. Acad. Sci. USA 71: 3400-3404 (1974)

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Siegel,M.I., McConnel1,R.T. and Cuatrecasas,P.: Aspirin-like drugs interfere with arachidonate metabolism by inhibition of the 12-hydroperoxy5,8,10,14-eicosatetraenoic acid. Proc. Natl. Acad. Sci. USA 76: 3774-3778 (1979)

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Editor:

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(1980)

G. Kaley

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Received:

4-25-84

Accepted:

12-13-84

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