Retinol induces platelet aggregation via activation of phospholipase A2

Vol. August

154,

No. 15,

3, 1988

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1988

RETINOL VIA

INDUCES ACTIVATION

PLATELET

1075-1080

AGGREGATION

OF PHOSPHOLIPASE

A2

Tohru Nakano, Kohji Hanasaki, Saichi Matsumoto and Hitoshi Arita*

Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku,

Received

June

20,

Osaka,553 Japan

1988

Summary: All-trans-retinol induced aggregation of rabbit platelets, and this effect could be inhibited by a cyclooxygenase inhibitor and a thromboxane A2 (TXA2) receptor antagonist, indicating an essential role for endogenously produced TXA2. We found a two-phase arachidonic acid release in retinol-stimulated platelets. The first phase was induced by the action of retinol alone and not inhibited by TXA2 receptor antagonist. The second phase was induced via synergistic action of retinol and initially generated small amount of TXA2, and was inhibited by the antagonist. Moreover, we discussed that the arachidonic acid release may be mediated by the action of phospholipase AZ. 0 1988Academic Press,Inc.

Many reports on the biological roles of retinol have recently revealed its effects on cellular differentiation,

maintenance of reproductive capacity and glycoconjugate

biosynthesis (1). Of the many roles of retinol, however, only its involvement in the visual cycle (2) and that in the glycosyl transfer reaction (3) have been clearly defined at the molecular level. Bangham et al. examined the effect of retinol and its analogues on the artificial membrane and found that retinol could penetrate and expand a monolayer of lecithin-cholesterol at an air-water interface with considerable molecular specificity (4). These data therefore suggest a preferential

mecha-

nism for the biological effects of retinol to exist on the membranes of the targeting cells. In order to ascertain this, we examined the action of retinol on rabbit platelets and found1 that it induced platelet aggregation via activation of phospholipase A:! with liberation of a large amount of thromboxane A2 (TXA2). This result suggested that the biological effects of retinol on other cell types may be coupled with those of some products in the arachidonic acid cascade.

* To whom all correspondence should be addressed. The abbreviation used is: TXAz(Bz), thromboxane A2 (B2).

1075

0006-291X/88 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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METHOD

Preparation of rabbit platelets -Freshly drawn rabbit blood was mixed with 0.15 volumes of acid citrate dextrose (85 mM trisodium citrate. 70 mM citric acid. and 110 mM glucose) and 0.5 pg/ml prostaglandin El, and platelet-rich plasma was obtained by centrifugation at 160 x g for 10 min. Platelets were sedimented at 1,200 x g for 15 min and resuspended at 2 x 109 cells/ml in resuspension buffer (137 mM NaCl, 2.7 mM KCl, 1.0 mM MgClz, 3.8 mM NaH2P04, 3.8 mM Hepes, 5.6 mM glucose, and 0.035% bovine serum albumin, pH 7.35). If necessary, platelets were labeled with 10 pCi/ml [sH]arachidonic acid for 2 h or with 1 pM FuraZAM for 30 min at room temperature in the presence of 0.5 pg/ml prostaglandin El. Platelets were sedimented onto 40% bovine serum albumin, isolated with a column of Sepharose 2B, and resuspended in the resuspension buffer at 5 x 108 cells/ml. CaC12 (1 mM) was added to the platelets 2 min before stimulation. Platelets were stimulated in NKK HEMA TRACER 1 (Nikoh Bioscience Co., Ltd., Tokyo) at 37”C, and the aggregation was monitored. Cytoplasmic Ca2+ concentration was measured by the -Ca2+ mobilization method of Grynkewicz et al. (5) using a CAF-100 Ca2+ analyzer (Japan Spectroscopic Co., Ltd., Tokyo). Arachidonic acid release and lipid degradation [sH]Arachidonic acid-labeled nlatelets were stimulated in NKK HEMA TRACER 1. Stimulation was stormed bv adding 4 volumes of chloroform/methanol (1:2, v/v), and lipids were extracted-by the method of Bligh and Dyer (6). 3H-Labeled eicosanoids were separated by thin layer chromatography according to the method of BertelC et al. (7). The area corresponding to eicosanoids containing arachidonic acid, TXBz, hydroxyeicosatetraenoic acid and hydroxyheptadecatrienoic acid was scraped off and the radioactivity was measured. Arachidonic acid metabolites other than these eicosanoids were scarcely detected. 3H-Labeled phospholipids were separated by thin layer chromatography by the method of Rauser et al. (8). Radioactivity in the areas of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol was measured. TXAz formation Platelets were stimulated as described and stopped by adding equal volumes of 10 mM EGTA and 50 pM indomethacin. Platelets were precipitated and TXBz, a stable metabolite of TXA2, in the supernatant was measured by TXBz radioimmunoassay kits. Materials Retinol and indomethacin were purchased from Sigma, St. Louis. Fura2-AM was from Dojin, Kumamoto, Japan. U46619 was from Upjohn. Kalamazoo. TXB2 radioimmunoassay kits were obtained from New England Nuclear, Boston. SQ29,548 was synthesized at Shionogi Research Laboratories.

As shown

RESULTS AND DISCUSSION in Fig. lA, retinol (20 pM) induced aggregation

of rabbit

platelets

after a lag period of about 1 min. The threshold concentration of retinol for the induction of aggregation was 5 to 10 pM, but 20 pM retinol was necessary to obtain constant results without release of lactate dehydrogenase. Indomethacin (10 pM), an inhibitor of cyclooxygenase, inhibited the retinol-induced aggregation, indicating that secondary produced cyclooxygenase products might be involved in retinolinduced platelet activation. Furthermore, 1 pM SQ29,548, a specific antagonist of TXA2 receptor (9), blocked the platelet response to retinol. Using FuraB-loaded platelets, the retinol-induced elevation of cytoplasmic Caz+ concentration was observed, which occurred concomitantly with platelet aggregation (data not shown). 1076

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SQ

Fig. 1, Aggregation of rabbit platelets stimulated by 20 pM retinol (A) or 1 pM U46619 (B). Platelets were stimulated at the arrow. Increase of light transmission represents aggregation. Vehicle (CONT), 10 pM indomethacin (IM) or 1 pM SQ29,!j48 (SQ) was added 2 min before stimulation.

Indomethacin and SQ29,548 also inhibited it. These results suggested that retinol initially promoted the TXA2 formation probably via arachidonic acid release, which was necessary to induce platelet aggregation as well as Ca2+ mobilization.

Figure

1B indicates that 1 pM U46619, an agonist of TXA2 receptor (lo), induced platelet aggregation which was not inhibited

by indomethacin but completely blocked by

SQ29,548. As expected, retinol induced TXA:! formation by rabbit platelets, which was completely inhibited by 10 PM indomethacin (Table 1). SQ29,548 also inhibited the TXA2 formation although,

unlike indomethacin,

it could not interfere

with the

pathway of TXA:! synthesis (9). Similar results were obtained on arachidonic acid release as shown in Table 2. Retinol-induced arachidonic acid release represented by [sH]eicosanoid formation was greatly reduced by inhibiting

TXA2 formation with

indomethacin and also by a blockade of TXA2 binding to the receptor with SQ29,548, although these compounds did not directly affect the arachidonic acid release (9,111. These results suggested that only a small amount of TXA2 was initially

generated

by retinol and it further provoked the formation of a greater amount of TXA2. Therefore, we examined whether U46619 would induce arachidonic

acid

formation. As shown in Fig. 2, U46619 alone did not induce [SHleicosanoid formation at concentrations of 10 to 1,000 nM but did induce full platelet aggregation at

Table 1. TXA2

Control + IM + SQ

formation

induced

by retinol

TXA2 (rig/ml) 362 + 45 l&l 18 f 3

Rabbit platelets were stimulated with 20 pM retinol for 3 min and TXBs, a stable metabolite ofTXA2, was measured. Vehicle (Control), 10 pM indomethacin (IM) or 1 uM SQ29,548 (SQ) was added 2 min before stimulation. The data are mean f S.D. (n = 3). 1077

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Table 2. [sH]Eicosanoids

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formation induced by retinol

[sH]Eicosanoids formation (dpm x 10-3) 31.1 + 3.7 4.9 k 0.8 3.7 k 1.1

Control + IM + SQ

[aH]Arachidonic acid-labeled platelets were stimulated with 20 FM retinol for 3 min. Increment of [3H]eicosanoids over the resting level is represented. Vehicle (Control), 10 PM indomethacin (IM) or 1 pM SQ29,548 (SQ) was added 2 min before stimulation. The data are mean f S. D. (n = 3).

1,000 nm (Fig. 1B). In order to clarify the induction

of arachidonic

the synergistic

acid release,

retinol

action of retinol

and U46619

were

and TXA2 for simultaneously

added to platelets in the presence of 10 PM indomethacin to avoid endogenous TXA2 formation. Retinol(20 pM) induced only a small amount of 13Hleicosanoid formation in the presence eicosanoid

of indomethacin.

formation

However, addition of U46619 induced [3Hlin a dose dependent manner (Fig. 2), indicating that retinol

and U46619 synergistically induced arachidonic acid release. Two pathways have been proposed for arachidonic acid release; phospholipase monoacylglycerol U46619

induces

A2 (12) and the second involves lipases activation

(13, 14).

and arachidonic with

Some observations

of phospholipase

U46619 does not enhance arachidonic Therefore, activation of phospholipase

phospholipase have

C in platelets

one involves

C and diacyldemonstrated (15-17).

and that

However,

acid release as shown in this study (Fig. 2). C may not lead to arachidonic acid release

acid seems to be released mainly

by the action of phospholipase

AZ.

In order to identify the source of arachidonic acid released by the stimulation retinol, the decrease in the 13Hlarachidonic acid-labeled phospholipids by the

stimulation was studied. phosphatidylethanolamine

As shown in Fig. 3, radioactivity in phosphatidylcholin, and phosphatidylinositol decreased by 15 to 20%,

whereas no decrease was detected for phosphatidylserine. This suggests that phospholipase A2 actually works to liberate arachidonic acid in retinol-stimulated platelets, because phospholipase C acts on only inositol phospholipids (18). In this study, we first demonstrated that retinol induced platelet activation

via

enhancement of arachidonic acid release. Cell differentiation is one of the wellknown biological effects of retinal(1). Some prostanoids have also been reported to promote cell differentiation (19, 20). These findings raise the possibility retinol-induced cell differentiation is mediated by arachidonic acid release

that and

prostanoid synthesis, although direct evidence is not available. We have already reported that collagen activates platelets in a manner similar to retinol (11, 21). It first generates a small amount of TXA2 and then induces a greater amount of arachidonic acid release synergistically with the initial TXA2. It 1078

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l+

Radioactivity 0

2

(“1. of control) 50

100

PC 1

PE

ow

p’ PS

/“0

10 30 U46619

100 3001000 (nM)

03

of synergistic action of U46619 and retinol for 13Hleicosanoids acid-labeled platelets were stimulated by U46619 (0) or U46619 plus 20 pM retinol (0) for 3 min in the presence of 10 pM indomethacin. Increment of [sH]eicosanoids over the resting level is represented. Fi . 3. Decrease of [sH]arachidonic acid-labeled phospholipids. IsHlArachidonic izEn- abeled platelets were stimulated by 20 pM retinol for 3 min. Radioactivity of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) is represented with the resting level as 100%. The data are mean k S.D. (n = 3).

is interest.ing that collagen and the smaller molecule retinol have similar effects on platelets. Although the mechanism of enhancement of phospholipase A2 action by these molecules remains obscure, more precise analysis of the interaction between these molecules and the platelet membrane may provide some answers.

We are grateful

ACKNOWLEDGEMENT to Mrs. A. Terawaki of our laboratories

for her skillful

technical assistance and also to Miss M. Katayama for typing this manuscript. wish to thank Dr. S. Hagishita for the synthesis of SQ29,548.

We

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