The role of zlpoE in inhibitory elects af apoE-rich HDL on plntelet function

Rcceivcd27 Dcccmbcr 1990;rcvired versionreceived I3 Febrtwy

1991

Apslipuprauln E- (ApoE-) rich high-density IipeproIcin (MDL), which WI preprrrd from rhc bound fracricm al’nunnalipcmic valun~ecrx an heparin&phuroae and from I hyprrlphrrlipclpreleincmic pn\icnt, peknrly inhibited ngyrcyatian of humnn pla~rlc~x In II, dose-dcpcndcnt rashinn. Dimyristoyl pkosplratidylcholinc lipusamr with epnE (apeEs DMPC) also inhibited plattlet aggrrgutiun, und incubation of washed phtclr~r with opoE* DMPC resulted in the relcn~c of cholesterol into the ruprnatant in II time* and dare-deprndcm mnnncr, Thrsc rcruhr xulg~cs( rhnt up& plny?l a major ralc in the inhibirary eiket of ~qx.&rich HBL in plrrrelct function, presumably due to the rclc~txr UF chnleatcrul from tha ph~smu mcmbranc. High-dcnrhy

lipoprotein; Chelcalcrel; fWclcr qgrcgtlrion;

1. INTRODUCTION Platelets play an important role in the formation of thrornbus and atherosclerotic lesions [ 11. Platelets from the patients withhypercholesterolemia show an increase in aggrcgability, especially for eoinephrine [2,3] and their plasma membranes contain a great deal of cholesterol, suggesting that the content of cholesterol in the plasma memhranc has a critical role in determining the sensitivity to stimuli. A number of reports have indicated that low-density lipoprotein (LDL) enhances agonist-induced platelet aggregation, whereas highdensity lipoprotein (HDL) has an opposite effect [4-91. Recently, Desai et al. [g] showed that one of the critical factors of HDL in reducing the aggregability was the content of apoE, because HDLz showed more potent inhibition than HDLs. However, the mechanism of potent inhibition by apoE-rich HDL remains unknown. In this report, we focus on the role of apoE in the inhibitory effects of apoE-rich HDL on platelet function and investigate the interaction of dimyristoyl phosphatidylcholine liposome with apoE (apoE*DMPC) with platelets, especially in terms of cholesterol exchange in the plasma membrane. 2. MATERIALS AND METHODS 2.I, Materials and proteins Dimyristoyl phosphatidylcholinc (DMPC) and luciferine-luciferase were purchased from Sigma (St. Louis, MO). cu-thrombin was purCorrespondence address: M. Higashira, First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-l Wongo, Bunkyo-ku, Tokyo 113, Japan

82

Apulipoprolcin

E

chased from Mochida Phsrmnccurical (Tokyo, hpan), Nn’“‘l was obtained from New England Nuclear (Bamn, MA), Rcrombinant apoE was a gift from Mitsubishi Karei (Kanagawa, Japan). Apolipoprotein A.1 (ApoA-I) was purified from normal plasma according to the method of Schonfcld CI al. [91. 1,3,3,4-telrachloro-3~~-diphenylglycouril (IODO-GEN) was purchased from Pierce (Rockford, IL). Hcparin-Scphnrosc was made according to the method of Iveriur ct al, [IO]. ConAGphnrosc was purchased from Pharmacia, Uppsnla, Sweden. 2.2. Isolation of pktsrnn lipoprofeins Human HDL (c-f= 1.063-I .21 g/ml) was isolated from plasma obtained from normolipemic fasted volunteers by ultracentrifugntion in a 50 Ti rotor (Hitachi) (71. ApoE-rich and apoE-poor HDL fractions of normal plasma were prepared by heparinSepharore column chromatography as described by Wcisgraber ct al. [l I], The ApoEi ApoA-1 ratio in apoE-rich HDL fraction was calculated to bc about 0.65. The ApoE-rich HDL fraction was also isolated from plasma of a hyperalphalipoproteinclnic patient with cholesteryl ester transfer protein deficiency. A fraction of d< 1,063 g/ml of the patient’s plasma was applied to the ConA-Sepharose column to remove apoB as described previously [l2), and flow-through fractions (apoE-rich HDL) were collected. The ApoE/ApoA-I ratio of this fraction was about 0.9. The ApoE’ DMPC complex or apoA-1’ DMPC complex was prepared by incubating DMPC with recombinant apoE or purified apoA-I overnight at room temperature with a lipoprotein/DMPC ratio of 1:3.75 (weight/weight) according to the method of Innerarity et al. [13]. Free apolipoprotein was removed by ultracentrifugation , The electron micrography of negatively stained apoE* DMPC was carried out according to the tnethod of Basu et al. [14]. 2,3, Iodination and binding experiments LDL was iodinated by using IODO-GEN [15]. The specific ac. tivities were 200-400 cpm/ng protein. [‘251]LDL binding was carried out at 37QC as described previously [7]. 2.4. Plrrteiet function Washed platelets were

prepared

Published

as described

previously

by EIsevier Science Publishers

[16].

B. V,

= 6.9) fran a I~yperalphalipopro~eInemie patient with plasma cholerteryl ester transfer pro-

CapaE/q~erA-f

deficiency on platelet qg,regation. This fraction also inhibited collagen- and thrombin-induced platelet aggregation in a do&-dependcnr fashion (Fig 2). HDL from normal donors at concentrations up to 0.2 mg protein/ml showed little inhibition (data kot shown), These results suglcst that apoE may have a criricsllrole in the potent inhibition of platelet function by HDLa, as suggested by Desai et al, [8I. In order to study the inhibitory effect of apoE on apsISrich HDL which also conrains other apolipopran tcins, i.e. apoA-I, we prepared liposomes consisting of only apoE and DMPC, which had disc forms like nascent HDL (Fig 3A). ApoE’ DMPC inhibited collagen-

tein

3. RESULTS AND DISCU%SIBN

We rcportcd prcviousiy (71 that HDLa inhibited collagen- and thrombin-induced platelet algregarion mom potenrly than HDLI\, Phesc results confirm the earlier results of Bcsai ct al. [S], who suggested that the more potent inhibition of ADP-induced aggregation by HDLz than that by HDL3 was derived from the difference in apoE conrcnr. ApoE4ch HDL (apoE/apoA-I = 0.65) from normal donors prepared by heparin-Sepharose column chroma-

Fig, i. inhibition of piatekt aggregation and ATP release by apoE-rich !-!DL. (A) Elution profile of heparin-Sepharose column. The concentration of protein in each fraction was measured by the method of Bradford [30]. (B,C,D) Inhibition of ADP- (B), collagen-(C), or thrombin- (D) induced platelet aggregation and ATP release by apoE-rich HDL. The values in parentheses are those of ATP released (uM). Agonists were added to washed platelets (30% lO’/,ul) preincubated with several protein concentrations of apoE-rich HDL (shown in figures) for 30 min at 37°C. LTU, light transmission unit.

83

Thrombin(U,

I

lU/d)

Pig. Z!, InhMian of csllnycn- (A) or thrombin* (8) induccB plntelct aygrsplion by rhr HDL haion from n trypcralphalipopretcincnrif prricnr. Aponivrls wore a&k : ro wsshcd plevAelJ (30X ICY/& preincubnrrtl with ncvcrrl protein conccnlraliOn9; or HDL ror 10 min 81 3W2 LTU, Lighr tliPlldlilil~i~n unit.

-_ Collagen

1’2pglmlj e

d

--

Thrombin

::

(0.1 U/ml)

40 Protein

Concentration

(llg/ml)

Fig. 3. Inhibition of [1251]LDL binding to platelets and platelet aggregation by apoE*DMPC. (A) Electron micrography of negatively stained apoE*DMPC. (B) Inhibition of [‘*511LDL binding by cold LDL (a), apoE*DMPC (8) or apoE alone (A), (C,D) Inhibition of collagen- (C); or thrombin- (D) induced platelet aggregation by several protein concentrations of HDL, (a) control; (b) apoA-l.DMPC, 100 &ml; (c,d,e) apoE* DMPC, 25 /rg/ml, 1OOyg:/ml and 200pg/ml, respectively. Experimental conditions were the same as in Fig. 1, LTU, light transmission unit.

84

wnd thrombin-jl~~ueed pIatelcr ~~~r~~~tion iin Al doledependent fashion {Fig, 367,D). fn contrast, few ina hlbirory cffeeca were seen with apoA-I- BMPC UFto 0, I m&/n& ApoE-DMBC gorenrly inhibited [I* I]LJX. binding to plrfelefa CR& X4). Scrrrehrrd Rnalysio reveal* cd ohnf the affinify was about 18 rknes that of native LDL (unpublished observation). Neither DMPC (data not shown) nor apoE alone (Fig 38) showed any inhibition, ~u~~~~~~~$ that apoE acquires the heility of inhibition by forming an a&e eomplcx with DMPC, Thus, platelct=typc LDL receptor [7,20] may nhare the cnpaeity of bindin to both apoF3 and apoE, like the elaxsieal LBL recepfor of fibroblarts [2L]. Why does apoE~ BMPC have inhibitory peopcrties? To obtain insight into ttw mechanism of this inhibition, the effects of apoEmDMPC on [Ce”“]i increase and aggregacion by rhrombin (0.1 U/ml) were examined for

n

”

30

0

Incubation

Fig, 4. Inhibition

60 Time

ol thronrbin-induced

inerrase in [CO’“)~ by

various incubation times. Borh the increase in [Ca’*]I and the ageregntion were inhibired by apoE#DMPC ClOO&ml) in a rime-dependent fashion (Fig 4), No in-

c

hibition was seen wifh a shorter incubation

tmtt-4

pla~clct ag~rc@cien

and the

npoE*i&WC. Washed platelet (30 x lO’/pl)

were pfcincubrued

nt 37% in the presence or ubacnec of apoE~ BMPC for various incubntion periods, and ~Rcn thrombin (0, I U/ml) was added. The extent of [Caa“]i ircrcase (0,01 and maximal slope(aggregation/min) (A, A) were calculs&d as pcrccnt of control. The vnlrres are means f SD. of 3 cxpcrirnents. apoA-I*DMPC (0, A); apuE*DMPC (0, A).

(I00&nrl)

DMPC

Concentration

time, i.e. 5

min (Fig. 4). Thus apoE*DMPC seetns to inhibit the thrombin-induced receptor-mediated signal transduction, by affecting the plasma membrane in a rimedependent fashion. We measured the amount of cholesterol in the supernatant during incubation of washed platelets with apoE* DMPC. ApoE*DMIJC released the cholesterol in the supernatant

in a dose- and time-dependent

fashion

OqYml~

5

5 52

u0

50

I

0

100 Protein

Concentration

200 (,,g/ml)

Time

(min)

Fig. 5. The release of cholesterol from platelet membrane by apoE+ DMPC. A. Cholesterol or LDH release as a function of lipoproteins of DMPC alone. Washed platelets (30 x lo’/& were preincubated with apoE* DMPC, Apbh-i*DkiPC or DMPC alone for 30 min al 37°C and then put on ice. The supernatant after centrifugation at 3000 rpm for 10 min was determined for cholesterol content or LDH release. Cholesterol release (0, A, 0); LDH release (e, A ,m); apoE.DMPC (0,8); apoA-l.DMPC (A, A); DMPC alone (0,~). The concentration of DMPC vesicles, 360 &ml, was the same concentration of DMPC that was present in the lOOpg/ml apoE*DMPC. B. Cholesterol or LDH release by apoE*DMPC (100 &ml) as a function of incubation time. Experimental conditions are the same as in A.

85

(Pips. SA,B), ApoE* IXWC: WI fi@ml) ~cleasccf 1Qa3% of ekslkatcroI Pbr 30 nin of incubrfIon (meank$D for J separafc cxpcrimenfs), tiftje cPfcef wtu seen with &her BMPC ~Isna m agoA- 1Bh4BC. LDH release was leas than 3% in (111samples (Fig. fA), The time dependencyof the extent ofcholercerol relers= ed by apoE 0DMPC was parallel with chat of the inhibiw tion of the hCaf*]l increase and the aggregation induced by chrombin (Figo 4 and 33). A longer ineubarian with

apoE’DMPC:(lQB/cg/ml), for 6 h, caused the apparent release of lSDH (Fig, SB), suggesting that removal of cholesterol From the plasma membrane lends fa fragility of platelet membrane. In 1975 Shartil et al. [22) showed that platelets incubated with cholesterol-rich liposomes [high eholesterol to phosphalipid ratio (C/PL)] had an increased sensitivity to epinephrine and ABP and that their platelets contain higher amounts of’ cholesterol, Platelets exposed to cholesterol-rich liposomes were found to have increased membrane microviscosity [23) and increased basal levels of adenylate cyclase [24], These observations in vitro may have a counterpart in human disease since plarclets from individuals with type ila hyperlipoproteinemia have an increase in membrane C/PC [25], and they are more sensitive than normal to aggregating agents [2,3]. More recent studies have indicated that platelets with decreased C/PL showed a decrease in the liberation of arachidonafe [26,27], the binding capacities of fhrombin [28] and the levels of fhromboxane RX formation [27,29]. Thus, the cholesterol content of the plasma membrane has been thought to have a crucial role in determining the sensitivities to agonists. This is the first report showing that DMPC liposomes with apoE release cholesterol from the platelet membrane, We here propose a hypothetical model: that apoE-rich HDL releases cholesterol from the plasma membrane of platelets, resulting in a decreased response to stimuli. However, since apoE*DMBC is a simple model for apoE-rich WDL and lacks other apolipoproteins, i.e. apoA-I as well as lipids like cholesterol and triglyceride, we cannot exclude the possibility that other inhibitory factors may lead to perturbation of the lipid-protein interaction in platelet membranes with different mechanisms, resulting in a decreased platelet function. Acknowledgements: We thank Miss Rika Hino and Miss Asami Tajimafor their excellent technical assistance. This work was suppork by a Grant-in-Aid for Scientific Research from the Ministry of Education,

Science and Culture of Japan.

[I] Row, R. (1984%N, EnpI, J, M*d, 3t4, 481-500.

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(t51 Markwclt,