Vol. 181, No. 3, 1991 December 31, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1331-1336
INHIBITION OF PLATELETFUNCTION BY RICE-DENSITYLIPOPROTEIN PROM A PATIENTWITHAPOLIPOPROTEIN II DEFICIENCY
Masaaki Higashihara”.
Makoto Kinoshita’.
Shoji Kume’.
Tario Teramoto’ and Kiyoshi Kurokawa’ 1 First Department of Internal Medicine. Fuculty of Medicine, University
of Tokyo. 7-3-l Bongo. Bunkyo-ku. Tokyo 113. Japan
a Central Clinical
Laboratory, YawanashiMedical College, Yaaanashi, Japan
Received November 12, 1991 SUMMARY: Apolipoprotein E-(apoE-) rich high-density lipoprotein (HDL) of normal subjects showedmarked inhibitory effects on platelet aggregation and ATP release as comparedwith apoE-poor HDL, suggesting that apoE has inhibitory effects on platelet function (Desai et al. J. Lipid Res.30:831. 1989; Higashihara et al. FEBSLett. 282:82, 1991). A patient with apoE deficiency showedevidence of decreased platelet aggregability in platelet-rich plasma, but normal aggregability in washedplatelets. Both patient’s plasma and HDLfraction inhibited platelet aggregation of normal subjects. Patient’s HDLreconstituted with recombinant apoE showedfurther inhibitory effects on platelet function. These results suggest that apoE is a potent, but not unique, inhibitory factor for HDL. 0 1991 Academic Press,
Inc.
Epidemiologic studies have shownan inverse association of highdensity lipoprotein
cholesterol
(HDL-C) levels and incidence and mortality
of ischemic heart disease (1.2). Although the underlying mechanismfor this inverse association is not fully
understood, it is proposed that HDL
promotes the removal of free chlesterol that HDLhas an inhibitory
from peripheral tissues (3-5) and
effect on platelet
function (6.7).
Platelets
play an important role in the genesis and subsequent development of atherosclerotic platelet
lesions (8). Thus, the inhibitory
effects of HDLon
aggregation and release reaction are thought to be one of the
anti-thrombotic
factois
in the plasma components.
* To whomcorrespondence should be addressed.
1331
0006-291X/91 $1.50 Copyright 0 I991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
181,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
ApoE is one of the major apolipoproteins lipoprotein
lipoproteins
inherited
showing
disease
atherosclerosis,
triglyceride
levels
(10).
Recently,
crucial
role
in the inhibitory
clarify
whether
factors
share these effects,
and
and characterized
(11.12)
inhibition
and we (13)
in inhibition
we studied
aggregation
constituent
of HDL on platelet
apoE is the only factor
and
of platelet
that apoE. a minor protein effects
is a rare
cholesterol
Desai et al.
HDL shows strong
suggesting
with
apoE deficiency
elevated
xanthomas.
and release,
lipoprotein
Familial
uptake of
type 111 hyperlipoproteinemia
by premature
that apoE-rich
(9).
COMMUNICATIONS
of very low-density
(VLDL). and appears to mediate the hepatic
triglyceride-rich
reported
RESEARCH
the effects
of HDL, has
function.
To
of HDL or other of low-density
(LDL) and HDL, which were prepared from plasma of a patient
apoE deficiency
(14).
on the platelet
functions
of both this
patient
normal subjects.
MATERIALS AND METEODS The clinical features and analysis of genetic defects in a patient with apoE deficiency (39-year-old man) were described previously (14). Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) from the patient were prepared as described previously (15). Plasma lipoproteins from normolipemic. fasting (12-16h) subjects and from this patient were isolated as described earlier (16). The following fractions of lipoproteins were obtained: LDL. d=1.019-1.050 g/ml; HDL2. d=l. 063-l. 125: HDLs. d=1.125-1.21. Concentrations of lipoproteins are given as their protein contents as determined by the method of Lowry et al. (17). Plasma lipid levels were determined by the enzymatic method as described previously (18). ApoE-rich HDL vas prepared on a heparin-Sepharose affinity column as reported previously (13). Recombinant apoE was a gift from Mitsubishi Kasei (Kanagawa, Japan). Reconstitution of apoE with HDL from the patient was carried out according to the method of lnnerarity et al., as described previously (13).
RESULTS AND DISCUSSION Several
reports
platelet
function
unknown.
Recently,
induced
platelet
HDL fractions
(7.8).
although
we reported aggregation
inhibited
dependent fashion, (13).
have proposed that HDL has inhibitory
These results
while
the
that HDLn inhibited more potently
platelet
collagen
on
remains and thrombin-
than HDLa. and that apoE-rich
aggregation
the effect
confirm
of inhibition
mechanism
effects
and ATP release
in a dose-
of apoE-poor HDL was not significant
the earlier
reports
1332 =
by Desai et al.
(11.12).
Vol.
BIOCHEMICAL
181, No. 3, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
and suggest that apoE has an important role in the inhibitory HDL. We further
showedthat liposomes constituted
phosphatidylcholine resulting
lipoproteins
of
with apoE and dimylistoyl
(apoE* DMPC)removed cholesterol from the membrane,
in decreased platelet
the functional
effect
function (13). To gain further
role of apoE on platelet
function,
from a patient with apoE deficiency.
plasma cholesterol and triglyceride
insight into
we prepared platelets The patient
and
showedhigh
levels, and poorly controlled diabetes
mellitus or heavy drinking worsened these levels. The patient’s
platelets
were prepared on separate days: one when he was in hospital and his diabetes mellitus was under good control (July 17. 1989). and the other whenhe was out of hospital and lacked good control over it (May 25, 1990). As shown in Fig. 1. PRP under both conditions showeddecreased responses to ADP (10 PM). collagen (2 fi g/mL), epinephrine (10 .uM) and ristocetin
(1.2-2.4
mg/mL),
although that under good control showeda more
greatly decreased response. Furthermore, the patient’s
platelets
resuspendedwith normal PPPshowednormal responses to these agonists (not shown). In contrast,
normal platelets
resuspendedwith patient’s
PPPshowed
decreased responses to these agonists (not shown). Gel-filtrated and washedplatelets
platelets
showednormal responses to collagen (2 ug/mL) or
012345
012345
Time (mid
Fig. 1. Platelet aggregation study of this patient. PRP was prepared two separate days and aggregationwas inducedby ADP(10 fiti)(
on
collagen
(2 fl s/ml)(B). epinephrine (10 flM)(C). or ristocetin (1. 2-2. 4 &ml)(D). a. PRPdated July 17. ‘1989; b, PRPdated May25. 1990;c.PRPof control. Closed arrow.
each reagent
added; open arrow,
final. 1333
ristocetin.
2.4 mg/nL in
Vol. 181, No. 3, 1991
m
BIOCHEMICAL
(0.1 U/m@
Thrombin
rP
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1331-1336
INHIBITION OF PLATELETFUNCTION BY RICE-DENSITYLIPOPROTEIN PROM A PATIENTWITHAPOLIPOPROTEIN II DEFICIENCY
Masaaki Higashihara”.
Makoto Kinoshita’.
Shoji Kume’.
Tario Teramoto’ and Kiyoshi Kurokawa’ 1 First Department of Internal Medicine. Fuculty of Medicine, University
of Tokyo. 7-3-l Bongo. Bunkyo-ku. Tokyo 113. Japan
a Central Clinical
Laboratory, YawanashiMedical College, Yaaanashi, Japan
Received November 12, 1991 SUMMARY: Apolipoprotein E-(apoE-) rich high-density lipoprotein (HDL) of normal subjects showedmarked inhibitory effects on platelet aggregation and ATP release as comparedwith apoE-poor HDL, suggesting that apoE has inhibitory effects on platelet function (Desai et al. J. Lipid Res.30:831. 1989; Higashihara et al. FEBSLett. 282:82, 1991). A patient with apoE deficiency showedevidence of decreased platelet aggregability in platelet-rich plasma, but normal aggregability in washedplatelets. Both patient’s plasma and HDLfraction inhibited platelet aggregation of normal subjects. Patient’s HDLreconstituted with recombinant apoE showedfurther inhibitory effects on platelet function. These results suggest that apoE is a potent, but not unique, inhibitory factor for HDL. 0 1991 Academic Press,
Inc.
Epidemiologic studies have shownan inverse association of highdensity lipoprotein
cholesterol
(HDL-C) levels and incidence and mortality
of ischemic heart disease (1.2). Although the underlying mechanismfor this inverse association is not fully
understood, it is proposed that HDL
promotes the removal of free chlesterol that HDLhas an inhibitory
from peripheral tissues (3-5) and
effect on platelet
function (6.7).
Platelets
play an important role in the genesis and subsequent development of atherosclerotic platelet
lesions (8). Thus, the inhibitory
effects of HDLon
aggregation and release reaction are thought to be one of the
anti-thrombotic
factois
in the plasma components.
* To whomcorrespondence should be addressed.
1331
0006-291X/91 $1.50 Copyright 0 I991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
181,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
ApoE is one of the major apolipoproteins lipoprotein
lipoproteins
inherited
showing
disease
atherosclerosis,
triglyceride
levels
(10).
Recently,
crucial
role
in the inhibitory
clarify
whether
factors
share these effects,
and
and characterized
(11.12)
inhibition
and we (13)
in inhibition
we studied
aggregation
constituent
of HDL on platelet
apoE is the only factor
and
of platelet
that apoE. a minor protein effects
is a rare
cholesterol
Desai et al.
HDL shows strong
suggesting
with
apoE deficiency
elevated
xanthomas.
and release,
lipoprotein
Familial
uptake of
type 111 hyperlipoproteinemia
by premature
that apoE-rich
(9).
COMMUNICATIONS
of very low-density
(VLDL). and appears to mediate the hepatic
triglyceride-rich
reported
RESEARCH
the effects
of HDL, has
function.
To
of HDL or other of low-density
(LDL) and HDL, which were prepared from plasma of a patient
apoE deficiency
(14).
on the platelet
functions
of both this
patient
normal subjects.
MATERIALS AND METEODS The clinical features and analysis of genetic defects in a patient with apoE deficiency (39-year-old man) were described previously (14). Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) from the patient were prepared as described previously (15). Plasma lipoproteins from normolipemic. fasting (12-16h) subjects and from this patient were isolated as described earlier (16). The following fractions of lipoproteins were obtained: LDL. d=1.019-1.050 g/ml; HDL2. d=l. 063-l. 125: HDLs. d=1.125-1.21. Concentrations of lipoproteins are given as their protein contents as determined by the method of Lowry et al. (17). Plasma lipid levels were determined by the enzymatic method as described previously (18). ApoE-rich HDL vas prepared on a heparin-Sepharose affinity column as reported previously (13). Recombinant apoE was a gift from Mitsubishi Kasei (Kanagawa, Japan). Reconstitution of apoE with HDL from the patient was carried out according to the method of lnnerarity et al., as described previously (13).
RESULTS AND DISCUSSION Several
reports
platelet
function
unknown.
Recently,
induced
platelet
HDL fractions
(7.8).
although
we reported aggregation
inhibited
dependent fashion, (13).
have proposed that HDL has inhibitory
These results
while
the
that HDLn inhibited more potently
platelet
collagen
on
remains and thrombin-
than HDLa. and that apoE-rich
aggregation
the effect
confirm
of inhibition
mechanism
effects
and ATP release
in a dose-
of apoE-poor HDL was not significant
the earlier
reports
1332 =
by Desai et al.
(11.12).
Vol.
BIOCHEMICAL
181, No. 3, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
and suggest that apoE has an important role in the inhibitory HDL. We further
showedthat liposomes constituted
phosphatidylcholine resulting
lipoproteins
of
with apoE and dimylistoyl
(apoE* DMPC)removed cholesterol from the membrane,
in decreased platelet
the functional
effect
function (13). To gain further
role of apoE on platelet
function,
from a patient with apoE deficiency.
plasma cholesterol and triglyceride
insight into
we prepared platelets The patient
and
showedhigh
levels, and poorly controlled diabetes
mellitus or heavy drinking worsened these levels. The patient’s
platelets
were prepared on separate days: one when he was in hospital and his diabetes mellitus was under good control (July 17. 1989). and the other whenhe was out of hospital and lacked good control over it (May 25, 1990). As shown in Fig. 1. PRP under both conditions showeddecreased responses to ADP (10 PM). collagen (2 fi g/mL), epinephrine (10 .uM) and ristocetin
(1.2-2.4
mg/mL),
although that under good control showeda more
greatly decreased response. Furthermore, the patient’s
platelets
resuspendedwith normal PPPshowednormal responses to these agonists (not shown). In contrast,
normal platelets
resuspendedwith patient’s
PPPshowed
decreased responses to these agonists (not shown). Gel-filtrated and washedplatelets
platelets
showednormal responses to collagen (2 ug/mL) or
012345
012345
Time (mid
Fig. 1. Platelet aggregation study of this patient. PRP was prepared two separate days and aggregationwas inducedby ADP(10 fiti)(
on
collagen
(2 fl s/ml)(B). epinephrine (10 flM)(C). or ristocetin (1. 2-2. 4 &ml)(D). a. PRPdated July 17. ‘1989; b, PRPdated May25. 1990;c.PRPof control. Closed arrow.
each reagent
added; open arrow,
final. 1333
ristocetin.
2.4 mg/nL in
Vol. 181, No. 3, 1991
m
BIOCHEMICAL
(0.1 U/m@
Thrombin
rP
