304
Defective Protein Phosphorylation Associated
With Hypofunctions in Stroke-prone
Spontaneously Hypertensive Rat Platelets
Takako Tomita, Masahiko Ikeda, Yasuhide Inoue,
Tomohiro Mitubori, and Keizo Umegaki
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The mechanism of platelet dysfunctions in stroke-prone spontaneously hypertensive rats
(SHRSP) was investigated. Platelet aggregation was inversely correlated with blood pressure or
heart weight/body weight ratios in various strains of spontaneously hypertensive rats (SHR),
indicating genetic defects. Thrombin-induced 47 kDa protein phosphorylation was markedly
reduced in platelets of SHRSP compared with that in Wistar-Kyoto (WKY) rat platelets,
accompanying reduced aggregation and secretion, but in 20 kDa protein phosphorylation was
unchanged. Ca2+ ionophore A23187-induced responses were also significantly decreased in
SHRSP, and the degrees of the changes were greater than those by thrombin. However,
12-O-tetradecanoylphorbol 13-acetate-induced responses in SHRSP were similar to those in
WKY rats, suggesting that protein kinase C activity and its substrate were normally present in
SHRSP platelets. Phosphatidylinositol content in platelets of SHRSP was 20% less than that in
WKY rat platelets, but the contents of other phospholipids, including phosphatidylinositol4-monophosphate and phosphatidylinositol- 4,5 -bisphosphates, were unaltered. Thrombininduced formation of diacylglycerols and phosphatidic acid did not differ from each other at the
low concentrations. In the absence of Ca , thrombin-induced responses occurred to a similar
degree in both platelets, whereas the enhancements by Ca2+ were much greater in WKY rats
than in SHRSP. These results suggested that defective Ca2* functions in receptor-mediated
activation of protein kinase C and postkinase-mediated events appear to be an underlying
mechanism for the hypofunctions in SHRSP platelets. (Hypertension 1989;14:304-315)
W
e have reported that aggregation and secretion in response to various proaggregating agents, including the Ca2+ ionophore A23187, are reduced with the development of
hypertension in stroke-prone spontaneously hypertensive rats (SHRSP) compared with that in agematched normotensive Wistar-Kyoto (WKY) rats.1
The abnormalities in platelets of SHRSP are not
secondary to hypertension,2 that is, they are not
consequent to the circulation of in vivo activated
exhausted platelets due to vascular injuries. Thus, a
prolonged hypotensive treatment of SHRSP did not
correct platelet abnormality.3 The influx of Ca2+ is
not impaired in SHRSP platelets despite some delay
From the University of Shizuoka School of Pharmaceutical
Sciences (T.T., M.I., Y.I., T.M.), Shizuoka, and the National
Institute of Nutrition (K.U.), Tokyo, Japan.
This study was partly carried out under the NIBB Cooperative
Research Program (84-133).
Address for correspondence: Dr. Takako Tomita, University
of Shizuoka School of Pharmaceutical Sciences, 395 Yada,
Shizuoka, Japan (422).
Received October 6, 1988; accepted April 12, 1989.
in the thrombin-induced rise of intracellular free
Ca2+ concentration ([Ca2+](),4 nor was the formation
of thrombin-induced thromboxane B2 interfered with
in SHRSP platelets.5 In the present study, protein
phosphorylation, which plays an important role in
platelet functions,6 was investigated in platelets of
SHRSP and WKY rats to clarify the underlying
mechanism of defective functions of platelets from
SHRSP. It was demonstrated that platelet aggregation was inversely correlated with blood pressure
and heart weight/body weight ratios in WKY rats, a
cross of SHRSP and WKY rats, SHR, and SHRSP
and that dysfunctions of platelets from SHRSP
were correlated to the reduced phosphorylation of
47 kDa protein (P47), which is a major substrate for
protein kinase C in platelets. Since the hypofunctions of SHRSP platelets can be observed when
stimulated with various aggregators, the defect seems
to be distal to receptor occupancy. In addition to
phospholipid contents, thrombin-induced activation
of phospholipase C and formation of diacylglycerols
were not apparently related to the hypofunctions
and defective P47 phosphorylation in SHRSP plate-
Tomita et al
Defective Platelet Protein Phosphoryiation
305
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lets, but the causative defect would be in the
impaired Ca2+ functions involved in the phosphoryiation of P47 and in the reactions leading to physiological responses.
I14C]Serotonin Secretion
The assay was performed by a modification of the
methods of Hofmann et al11 and Holmsen and
Dangelmeier12 as described previously.2
Materials and Methods
Reagents
[ 1 4 C ] S e r o t o n i n (58.5 m C i / m m o l ) and
3
[ H]arachidonic acid (83.3 Ci/mmol) were obtained
from New England Nuclear (Boston, Massachusetts); [32P]orthophosphate was obtained from Japan
Isotope Co. (Tokyo, Japan); 12-O-tetradecanoylphorbol 13-acetate (TPA) was from Sigma Chemical
Company (St. Louis, Missouri); Ca2+ ionophore
A23187 was from Calbiochem (San Diego, California); thrombin was from Midori Cross Co. (Osaka,
Japan); x-ray film was from Fuji film (Kanagawa,
Japan); acrylamide monomer, AWV'-methylenebis-acrylamide, sodium dodecyl sulfate (SDS),
JV,iV,Af',Ar'-tetramethylenediamine, and Coomassie
brilliant blue R250 were from Nakarai Chemicals
(Kyoto, Japan); molecular weight (MW) protein
standard for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was from Bio-Rad Laboratories
(Richmond, California); standard phosphatidylinositol (soybean), PIP (bovine brain), PIP2 (bovine
brain), and phosphatidic acid (PA) were from Sigma
Chemical Company (St. Louis, Missouri); 1,2diolein was from Funakoshi (Tokyo, Japan); highperformance, thin-layer chromatography (HPTLC)
plates (Art 5641, 10x20 cm) and thin-layer chromatography (TLC) plates (Art 5721, 20x20 cm) were
from Merck (Darmstadt, FRG).
Analysis of Platelet Protein Phosphoryiation
The platelet protein phosphoryiation experiment
was performed principally by the method of Feinstein et al.10 Washed platelets, prepared according
to the method of Feinstein et al10 and resuspended
in buffer A (137 mM NaCl, 5.4 mM KC1, 0.2%
glucose, 25 mM Tris-HCl, pH 7.4), were incubated
with f^Jorthophosphate (0.2 mCi carrier free to
2x10'cells/ml) for 90 minutes at room temperature.
Phosphorus-32-prelabeled platelets (0.2 ml, 5x10*
cells/ml) were washed twice and then were incubated with stimulants (0.02 ml) in the presence of
1.5 mM Ca2+ in aggregometer cuvettes at 37° C
while being stirred (1,000 rpm). The reaction was
stopped by an addition of the SDS-stopping solution
(9% SDS, 6% mercaptoethanol, 15% glycerol, 186
mM Tris-HCl, pH 8.6 with a small amount of
bromophenol blue), and the mixture was boiled for
2 minutes. The protein was subjected to SDSPAGE by the method of Laemmlis.13 The stacking
gel consisted of 3% acrylamide, and the separating
gel consisted of 6-18% linear gradient acrylamide.
The gels were stained with Coomassie brilliant blue
and dried on filter paper. They were exposed to an
x-ray film to prepare an autoradiograph. Phosphorus32 content of the protein bands were analyzed by
densitometry of autoradiograph (Chromoscan 200,
Joice Loebie, Gateshead, England). Results were
expressed as amount of protein phosphoryiation
relative to unstimulated controls. Molecular weights
were determined using the following proteins as
standard: lysozyme 14,400, soybean trypsin inhibitor 21,500, carbonic anhydrase 31,000, ovalbumin
45,500, bovine serum albumin (BSA) 66,200, and
phosphorylase B 92,500.
Experimental Rats and Measurement
of Blood Pressure
SHR, SHRSP, and WKY rats were originally
provided by Professor K. Okamoto of Kinki University Medical School and have been maintained
by brother-sister breeding in our laboratory. Rats
had free access to laboratory chow (MF, Oriental
Yeast Co., Tokyo, Japan) and water. Male SHR
and SHRSP, aged 10-17 weeks unless otherwise
described, and age-matched male WKY rats were
used throughout this study. Mean blood pressure
was measured by a tail-pulse pick-up method7 in
unanesthetized rats after warming the body about
10 minutes in a chamber maintained at 37° C.
Preparation of Washed Platelets and
Measurement of Platelet Aggregation
Washed platelets were prepared as previously
described,8 principally according to the method of
Baenziger and Majerus9 or according to the method
of Feinstein et al.10 Platelet aggregation was measured in the presence of 1.5 mM Ca2+ at 37° C by a
turbidimetric method that used a four-channeled
NKK Hematracer 1 (Niko Bioscience, Tokyo,
Japan) as described elsewhere.8
Analysis of Platelet Phospholipids
To 1 ml washed platelet suspension (5x10* cells/
ml), 3.75 ml CHCl3:MeOH (1:2) was added. The
mixture was left overnight at 4° C, shaken vigorously for 1 minute with 1.25 ml CHC13 and 1.25 ml
2 M KC1 solution containing 5 mM EDTA, and
centrifuged for 20 minutes at 3,000 rpm.14 The
extracted phospholipids were resolved on a HPTLC
plate as described previously.8 Phospholipids were
eluted from silica with MeOH and analyzed for
phosphorus according to the method of Bartlett.15
Analysis of Platelet Inositol Phospholipids
Inositol phospholipids were extracted by the
method of Rittenhouse16 and resolved on a HPTLC
plate precoated with potassium oxalate.17 The inositol phospholipids were extracted from silica by
shaking with 3 ml CHCl 3 :Me0H:2 M HC1
(25:15:10) for phosphatidylinositols,18 and 3 ml
306
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Vol 14, No 3, September 1989
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Blood prassura (mmHg)
4jO
4JB
00 ( 101 )
Haart walght/Body weight
FIGURE 1. Inverse correlation between platelet aggregation and blood pressure (Panel a) and between platelet
aggregation and heart weight/body weight ratios (Panel b). Washed platelets from SHRSP (•), SHR (A), offspring of
WKYrats and SHRSP (A), and WKY rats (•) at 18-20 weeks of age were stimulated with thrombin (0.22 units/ml) for
3 minutes at 37° C in the presence of 1.5 mM Ca2+. WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats;
SHRSP, stroke-prone spontaneously hypertensive rats.
:
CHCl
MeOH:2.5 M HC1 (7:14:1.4) for PIP and
16 3
PIP2 and assayed
for phosphorus by the method of
Hess and Den.19
Measurement of Diacylgtycerol Formation
Platelet rich plasma (9 vol) was incubated with
[3H]arachidonic acid (8.33 AiCi/ml in 10% BSA, 1
vol) for 60 minutes at 37° C.20 Washed platelets
labeled with [3H]arachidonic acid (0.9 ml), which
were resuspended in buffer B (25 mM Tris-HCl, 137
mM NaCl, 5.4 mM KC1, 0.2% dextrose, pH 7.4,
6x10" cells/ml), were incubated with thrombin at
37° C in the presence of 1.5 mM CaCl2. Diacylglycerol was extracted and resolved by the method of
Rittenhouse.20
Measurement of Phosphatidic Acid Formation
Washed platelets (about 2x 109 cells/ml) were incubated for 90 minutes at 37° C in equal volumes of
neutralized [32P]orthophosphate (0.5 mCi/ml), and
washed phosphorus-32-labeled platelets were resuspended in buffer B (about 4x 108 cells/ml). Phosphorus32-labeled platelets were stimulated with thrombin
(20 fjd) in the presence of 1.5 mM CaCl2 (total volume
0.5 ml) at 37° C. PA formed was extracted and
resolved by the method of Kennerly et al.21
Assay of Protein
Platelet protein was measured by the method of
Lowry et al.22 BSA was used as the standard.
Results
Correlation Between Blood Pressure
and Platelet Aggregation
Hypofunction of platelets in SHRSP was not
secondary to circulation of degranulated platelets,
which was observed in deoxycorticosterone acetate
salt and renal hypertension,23 nor could it be recovered by a long-term duration of hypotensive
treatment.3 To confirm whether the hypofunctions
of platelets are due to genetic defects, the correlation of platelet aggregation and blood pressure was
examined using SHR, SHRSP, WKY rats, and a
cross from female SHRSP and male WKY rats at
18-20 weeks of age. These ages, a little older than
other experiments, were chosen for this experiment
because SHR develop high blood pressure more
slowly than SHRSP. The blood pressure of the
cross from SHRSP and WKY rats was similar to the
range found in SHR. As Figure 1A shows, there
was a strong inverse correlation between platelet
aggregation and blood pressure (r= -0.783). An
analogous inverse correlation (r= -0.744) was
observed between platelet aggregation and heart
weight/body weight ratio (Figure IB). Together
with other evidence,3-23 these results indicate that
genetic factors are involved in the observed dysfunctions of platelets in SHRSP.
Thrombin-Induced Protein Phosphorylation in
Stroke-Prone Spontaneously Hypertensive Rat
and Wistar-Kyoto Rat Platelets
Since protein phosphorylation in platelets precedes various physiological responses,6 numerous
reports have been presented on protein phosphorylation of human platelets. Before investigation on
protein phosphorylation of platelets from SHRSP
and WKY rats could be undertaken, we performed
comparative studies of protein phosphorylation
between human and rat platelets using the method
applied on human platelets by Feinstein et aJ.10 In
tomita et al
a)
Defective Platelet Protein Phosphoryiation
307
b)
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20K-
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W
w
180
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47K-
20K-
.
:
*
FIGURE 2. Sodium dodecyl sidfate-pofyacrylamide gel electrophoresis of platelets
labeled with nP from stroke-prone spontaneously hypertensive rats (SHRSP) and
Wistar-Kyoto
(WKY) rats. Panel a:
Coomassie blue staining from control platelets. Panel b and Panel c: autoradiographs.
Platelets labeled with nP from SHRSP and
WKY rats at 16 weeks of age were stimulated for 30, 60, 120, and 180 seconds with
thrombin (0.15 units/ml) in Panel b; in Panel
c they were stimulated for 30, 60, and 180
seconds with Ca2+ ionophore A23187 (0.5
fiAf). Molecular weights were determined
using the following proteins as standard:
tysozyme 14,400, soybean trypsin inhibitor
21,500, carbonic anhydrase 31,000, ovalbumin 45,500, bovine serum albumin 66,200,
and phosphorylase B 92,500. W, WistarKyoto rats; S, stroke-prone spontaneously
hypertensive rats; 47K, 47 kDa protein;
20K, 20 kDa protein.
•
platelets stimulated with thrombin (0.15 units/ml)
for 1 minute at 37° C, P47 was strongly phosphorylated in both human and rat platelets. Twenty
kilodalton protein (P20) phosphorylation was already
observed in unstimulated rat platelets, and the
change was only slight after stimulation whereas in
human platelets phosphorylation of P20 occurred
only on stimulation.
Figure 2 shows SDS-PAGE of phosphorus32-labeled platelets from SHRSP and WKY rats
308
Hypertension Vol 14, No 3, September 1989
80
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Tim. (mln)
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1 X 3
Tim* (mln)
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0J
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FIGURE 3. Thrombin-induced ['*C]serotonin secretion, aggregation, and protein phosphorylation in platelets from
SHRSP and WKY rats. Panel a: time courses. Washed platelets (5x10" cells/ml) from SHRSP ( • ) and WKY rats
(~o—), prelabeled with 32P as described in text, were stimulated for 30, 60, 120, and 180 seconds with 0.15 units/ml of
thrombin in the aggregometer tubes at 37° C. [NCjserotonin prelabeled platelets (4X109 cells/ml) were stimulated with
0.30 units/ml thrombin. Results are expressed as mean±SD (n=4 in protein phosphorylation, n=3 in [14C]serotonin
secretion). Panel b: concentration dependency curves. Platelets prelabeled with nP or [14C]serotoninfrom SHRSP (—6)—)
and WKY rats (--O-) were stimulated with thrombin (0.11, 0.22, 0.30, and 0.43 units/ml) for 3 minutes. Results are
expressed as mean±SD (n=4 in ["CJserotonin secretion, n=5 in aggregation) and mean of duplicates in protein
phosphorylation. SHRSP, stroke-prone spontaneously hypertensive rats; WKY, Wistar-Kyoto rats; P47, 47 kDa protein;
P20, 20 kDa protein.
when stimulated with thrombin (0.15 units/ml) for
30, 60,120, and 180 seconds. Figure 3 illustrates the
time courses and concentration-dependent curves
of thrombin-stimulated [14C]serotonin secretion and
aggregation and P47 and P20 protein phosphorylation.
Aggregation responses were always measured with
phosphorus-32-labeled platelets, prepared for protein
phosphorylation, to confirm no artificial alteration
during the labeling procedure. When phosphorus32-labeled platelets (5X108 cells/ml) were stimulated
with thrombin (0.15 units/ml) for various times at
37° C, P47 phosphorylation occurred instantly and
reached a maximum at 30 seconds. The time course of
phosphorylation was parallel to that of [uC]serotonin
secretion. In Figure 3B, showing concentrationdependent curves, platelets were stimulated with
thrombin (0.11, 0.22, 0.30, and 0.43 units/ml) for 3
minutes. Phosphorylation curve of P47 was consistent
with aggregation and secretion curves. Phosphorylation of P20 was not concentration dependent.
Ca2+ Ionophore A23187-Induced
Protein Phosphorylation
In human platelets stimulated with thrombin or
other physiological stimuli, there was a rapid and
transient rise in diacylglycerols and [Ca2+]i after the
hydrolysis of phosphatidylinositols.6 These two second messengers synergistically activate protein
kinase C whose substrate is P47. The Ca2+ ionophore A23187 and several phorbol esters such as
TPA mimic endogenous Ca2+ and diacylgrycerol
signals, respectively, bypassing receptors. To examine separately the two signals given by thrombin,
the Ca2+ ionophore A23187 was employed for the
survey of calcium pathway. Effects of the Ca2+
ionophore A23187 on platelet functions and protein
phosphorylation were comparatively shown in platelets of SHRSP and WKY rats (Figure 4). The Ca2+
ionophore A23187 (0.5 fiM) induced [MC]serotonin
secretion and aggregation in platelets of SHRSP to
remarkably low degrees compared with those in
Tomita et al Defective Platelet Protein Phosphorylation
p—-
309
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1
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b)
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0
0.1
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M
M
lonophor* A13187 {ptt)
0 0J
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lonophor* A111B7
0.4
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0.1
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OJ
0.4
loaophor* A11187 (pM)
0
0.1 OJ OJ 0.4
lonophor* A111B7 (||U)
4. Ca2+ ionophore A23187-induced [uC]serotonin secretion, aggregation, and P47 and P20phosphorylation in
platelets from SHRSP and WKYrats. Panel a: time courses. Washed platelets (5x10" cells/ml) from SHRSP (—•—) and
WKYrats (--O--) prelabeled either with nP or [NC]serotonin, were stimulated for 30, 60, and 180 seconds with 0.5 \xM
Ca2+ ionophore A23187 in the aggregometer tubes at 37° C. Results are expressed as mean of triplicates. Panel b:
concentration dependency curves. Platelets prelabeled with nP or [I4C]serotonin from SHRSP (—•—) and WKY rats
(~O~) were stimulated with varying concentrations of Ca2* ionophore A23187, for 3 minutes (secretion), 5 minutes
(aggregation), and 1 minute (protein phosphorylation). Separate points show aggregation responses to 1 minute of
stimulation. Results are expressed as mean±SD (n=5 in [I4C]serotonin secretion and in aggregation) and mean of
duplicates (protein phosphorylation and 1 minute aggregation). SHRSP, stroke-prone spontaneously hypertensive rats;
WKY, Wistar-Kyoto rats; P47, 47 kDa protein; P20, 20 kDa protein.
FIGURE
WKY rat platelets. SDS-PAGE of phosphorus32-labeled platelets from SHRSP and WKY rats,
when stimulated with the Ca2+ ionophore A23187
(0.5 IJM), was shown in Figure 2C. P47 phosphorylation in platelets of SHRSP was about one third of
that in WKY rat platelets. P47 phosphorylation
reached a maximum at 30 seconds and then gradual
dephosphorylation was observed. The degree of
differences in secretion, aggregation, and P47 phosphorylation between platelets of SHRSP and WKY
rats was much greater on Ca2+ ionophore A23187
stimulation (Figure 4A) than on thrombin stimulation (Figure 3A). Figure 4B shows concentration
dependency curves of the Ca2+ ionophore A23187
when stimulated for 1 minute for protein phosphorylation, 3 minutes for [14C]serotonin secretion, and
5 minutes for aggregation. Separated points in aggregation curves represent responses at 1 minute,
which were scarcely observed at lower concentrations. [14C]Serotonin release (at 3 minutes) induced
by 0.10, 0.25, and 0.35 uM Ca2+ ionophore A23187
was significantly higher in platelets of WKY rats
than in those of SHRSP. Aggregation responses at 1
minute were scarcely observed with Ca2+ ionophore
A23187 (0.4 uM) in platelets of SHRSP. At 1
minute, WKY 2+
rat platelets aggregated in response
to 0.4 ^.M Ca ionophore A23187. Aggregation
induced by 5-minute stimulation with Ca2+ ionophore A23187 (0.2, 0.3, and 0.4 uM) was also much
rat platelets than in platelets of
greater in WKY
SHRSP. Ca2+ ionophore A23187 did not induce
protein phosphorylation at less than 0.2 jiM, but at
0.4 fiM, P47 and P20 protein phosphorylation was
observed, and responses to it were greater in platelets of WKY rats than in those of SHRSP. The fact
that the magnitude of differences in various
responses to Ca2+ ionophore A23187 between platelets of WKY rats and SHRSP was larger than in
responses to thrombin may imply that calcium
functions are impaired in platelets of SHRSP.
12-O-Tetradecanoylphorbol 13-Acetate-Induced
Protein Phosphorylation
Although the magnitude of the difference between
the two strains varies depending on the stimulants,
310
Hypertension
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Vol 14, No 3, September 1989
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60
TPA (nil)
SO
0
20
40
60
TPA (nM)
«0
20
40
60
TPA (nH)
FIGURE 5. TPA-induced [HC]serotonin secretion, aggregation, and protein phosphorylation in platelets from SHRSP
and WKYrats. Panel a: time courses. Platelets prelabeled with "P or ["Cjserotonin from SHRSP {—•—) and WKYrats
("O") were stimulated with TPA (80 nM for aggregation and protein phosphorylation, 100 nM for [u'Cjserotonin
secretion) for 30, 60, 180, and 300 seconds in the aggregometer tubes at 37 ° C. Panel b: concentration dependency curves
of platelets prelabeled with 32P or ["Cjserotonin from SHRSP (—•—) and WKYrats (~O~) were stimulated with varying
concentrations of TPA for 5 minutes (['''Cjserotonin secretion and aggregation) and 1 minute (protein phosphorylation).
Each point and vertical bar represent mean±SD fn=J). TPA, 12-O-tetradecanoylphorbol 13-acetate; SHRSP, strokeprone spontaneously hypertensive rats; WKY, Wistar-Kyoto rats; P47, 47 kDa protein; P20, 20 kDa protein.
the significant reduction of thrombin- and Ca2+
ionophore A23187-induced P47 phosphorylation, as
well as [14C]serotonin secretion and aggregation in
platelets of SHRSP, suggests that protein kinase C
per se, or receptor-mediated activation of protein
kinase C in SHRSP, seems to be defective. As TPA
directly activates Ca2+-activated, phospholipiddependent protein kinase C,24 platelets from SHRSP
and WKY rats were stimulated with TPA (Figure
5). TPA (80 nM) gradually induced [14C]serotonin
secretion and aggregation without differences in the
magnitude of responses between platelets of SHRSP
and WKY rats (Figure 5A). Phosphorylation of P47
by TPA (80 nM) was peaked rapidly in 30 seconds.
In contrast, P20 was phosphorylated slightly and
slowly, which coincided with the study by Naka et
al.25 Figure 5B shows platelet responses to varying
concentrations of TPA. Five-minute stimulation
with TPA-induced secretion and aggregation
responses in a concentration-dependent manner;
the curves are similar in platelets of WKY rats and
SHRSP. One-minute stimulation with 10 nM TPA
fully phosphorylated P47. Higher concentrations of
TPA produced analogous results. TPA effect on P47
phosphorylation was seen at a concentration as low
as 0.5 nM (data not shown). Again, platelets of
SHRSP and WKY rats equally phosphorylated at
P47. Phosphorylation of P20 was not evident. These
results strongly indicate that protein kinase C activity and its substrate P47 were normally present in
platelets of SHRSP and that the defective protein
phosphorylation would be ascribed to incomplete
receptor-mediated activation of protein kinase C.
Phospholipid Contents in Platelets From
Stroke-prone Spontaneously Hypertensive
Rats and Wistar-Kyoto Rats
Phospholipids extracted from platelets from
SHRSP and WKY rats were resolved by TLC, and
the phospholipid composition was compared
between the platelets from the two strains. The total
lipid phosphorus (mean±SD) was 8.67±1.71 (n = 18)
and 8.92±1.65 (n=17) fig phosphorus/mg platelet
protein in SHRSP and WKY rats, respectively,
which were not significantly different from each
other. Although phosphatidylinositol is a minor
phospholipid in the platelet membrane, the content
in platelets of SHRSP was significantly lower by
Tomita et al
TPA
(80nM)
Defective Platelet Protein Phosphorylation
311
lonophora A23187
(O.SpH)
_ 80
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40
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BO
s
Tlrea (mln)
a
lonophora
TPA
A23187
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(O.OBpM)
1
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lonopbora
A23187
(O.OSpU)
40
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1
3
Tlma (mln)
6. Effects of TPA and Ca2+ ionophore A23187 on aggregation of platelets
from SHRSP and WKY rats. Platelets
from SHRSP {—•—) and WKY rats (-O-)
were stimulated with TPA or Ca2+ ionophore A23187 at the concentrations indicated in the figure. Each point represents
mean of triplicate assays. TPA, 12-Otetradecanoylphorbol 13-acetate; SHRSP,
stroke-prone spontaneously hypertensive
rats; WKY, Wistar-Kyoto rats.
FIGURE
TPA
+ (lOnU)
5
approximately 20% than that in WKY rat platelets.
Despite a significant difference of phosphatidylinositol content between platelets of SHRSP and WKY
rats (nmol/mg protein, SHRSP, 8.27±1.33 [n= 9];
WKY rats 10.60±1.59 [n=9], p<0.05), the PIP
content (SHRSP, 1.45 + 0.30 [n = 14]; WKY,
1.45+0.29 [n = 15]) and PIP2 content (SHRSP,
1.00±0.15 [n=18]; WKY rats, 0.93±0.16 [n = 18])
did not differ from each other. There was no difference in the two major phospholipids content: phosphatidylcholine and phosphatidylethanolamine.
Thrombin-Stimulated Diacylglycerol Formation
[3H]Arachidonic acid-labeled platelets were stimulated with thrombin to examine the formation of
diacylglycerol. Diacylglycerol was extracted from
platelets and resolved on TLC as described under
Materials and Methods. Diacylgycerol formation in
human platelets peaked at 15 seconds and then
decreased. However, in rat (WKY) platelets, the
formation of diacylglycerol peaked at 15 seconds as
in human platelets, then decreased slightly at 30
seconds, gradually increasing afterward. The formation in response to various concentrations of thrombin was examined simultaneously with aggregation
tracings. With lower concentrations of thrombin
(less than 0.15 units/ml), no significant formation of
diacylglycerol was observed in both platelets,
although aggregation responses were different
(SHRSP 31%, WKY rats 68%). In supramaximal
doses of thrombin, which induced full aggregation
in both platelets (SHRSP 80%, WKY rats 80%, no
significance), diacylglycerol formation was apparently observed; it was lower in platelets of SHRSP
than in WKY rat platelets. This diminished formation of diacylglycerol apparently reflects the lower
content of phosphatidylinositol in platelets of
SHRSP. Thus, it appears likely that reduced diacylglycerol formation from inositol phospholipids is
not responsible for attenuated responses of thrombininduced aggregation, secretion, and protein phosphorylation in platelets of SHRSP.
Thrombin-Induced Phosphatidic Acid Formation
To confirm that the initial step of platelet activation is not impaired, PA formation was examined in
platelets of SHRSP and WKY rats. As diacylglycerol produced from inositol phospholipids is quickly
transformed to PA, the formation of PA reportedly
reflects in vivo phospholipase C activity.26 Platelets
were labeled with [ P] and rinsed. There was no
difference in [32P] uptake into platelets between the
two strains. When phosphorus-32-prelabeled platelets were stimulated with thrombin (0.1 units/ml) for
various times, PA formation increased linearly with
similar rates in both platelets for 30 seconds, but
afterward the rate became slightly lower in platelets
of SHRSP than in those of WKY rats (16% less at 90
seconds in platelets of SHRSP compared with WKY
rat platelets without significant difference). The
concentration dependency of PA formation on stimulation (30 seconds) was examined simultaneously
with aggregation tracings. There was no difference
in [32P]PA formation between WKY rats and SHRSP
at the examined concentrations of thrombin despite
the significant difference in the aggregation response
at the concentration of 0.13 units/ml thrombin
(SHRSP 25%, WKY rats 86%). Thus, these results
suggest that neither phospholipase C activity nor
diacylglycerol formation are related to the defective
protein phosphorylation in platelets of SHRSP.
Effect of 12-O-Tetradecanoylphorbol 13-Acetate
and Ca Ionophore A23187 on Aggregation
Figure 6 shows the synergistic effects of TPA and
the Ca2+ ionophore A23187 on aggregation of platelets. As shown before, TPA (80 nM) induced aggregation similarly in platelets of both strains of rats,
312
Hypertension Vol 14, No 3, September 1989
TABLE 1. Effects of 12-0-Tetradecanoylphorbol and CaJ+ lonophore A23187 on Aggregation and Protein Phosphorylation in Platelets From SHRSP Stroke-prone Spontaneously Hypertensive Rats and Wlstar-Kyoto Rats
Ca
Protein
phosphorylation
(relative to control)
2+
ionophore
A23187
(MM)
0
0.1
Aggregation
(%)
TPA
(nM)
0
0
0
SHRSP
WKY
0
0
0
0
1
0.%
1.2
1
0
20
SHRSP
WKY
0.1
10
0.1
20
SHRSP
WKY
SHRSP
WKY
20
SHRSP
WKY
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0.2
1
1
0.4
0.4
WKY
10
0
P20
0
SHRSP
WKY
SHRSP
WKY
0.2
P47
1
1
0
SHRSP
0
0
0
0
2
trace
12
trace
27
trace
12.6
13.0
9.9
10.9
12.6
12.0
12.4
13.5
13.8
12.0
0.19
0.72
0.62
0.69
0.61
0.94
1.27
1.63
1.57
1.60
1.77
1.77
Platelets prelabeled with 32P from SHRSP and WKY rats were stimulated with TPA or Ca2+ ionophore A23187 for
1 minute at 37° C. Each value represents mean of triplicate determinations. TPA, 12-O-tetradecanoylphorbol
13-acetate; P47, 47 kDa protein; P20, 20 kDa protein; WKY, Wistar-Kyoto rats; SHRSP, stroke-prone spontaneousry hypertensive rats.
but the response to Ca2+ ionophore A23187 was less
in SHRSP than WKY rats (Figure 6, upper two
figures). Subminimum doses of TPA (10 nM) or
Ca2+ ionophore A23187 (0.05 jtM) did not cause
aggregation in both platelets (Figure 6, lower left).
However, co-stimulation with TPA and Ca2+ ionophore A23187 in the subminimum concentrations
induced aggregation; the responses were less in
platelets of SHRSP than WKY rat platelets (Figure
6, lower right).
Effect of 12-O-Tetradecanoylphorbol 13-Acetate
and Ca Ionophore A23187 on Protein
Phosphorylation
Platelets prelabeled with 32P from SHRSP and
WKY rats were stimulated with suboptimum doses
of TPA (10-20 nM) or Ca2+ ionophore A23187
(0.1-0.2 /j.M) for 1 minute. Platelet aggregation and
protein phosphorylation in response to these stimulations are shown in Table 1. Independent stimulation with Ca2+ ionophore A23187 (0.1-0.2 fiM)
induced neither aggregation nor phosphorylation,
whereas stimulation with TPA (10-20 nM) induced
full phosphorylation of P47 equally in platelets from
SHRSP and WKY rats without inducing aggregation. However, TPA (10-20 nM) and Ca2+ ionophore A23187 (0.1-0.2 ^M) together induced both
aggregation and phosphorylation of P47. Aggregation was less in SHRSP than in WKY rats, and the
phosphorylation was similar in SHRSP and WKY
rats. Thus, it is suggested that in platelets of SHRSP,
Ca2+ does not function normally, first in activation
of protein kinase C and second in the steps coupling
protein phosphorylation to physiological responses.
Ca2+ Dependency of Secretion, Aggregation, and
Protein Phosphorylation in Stroke-prone
Spontaneously Hypertensive Rat and
Wistar-Kyoto Rat Platelets
Effects of extracellular Ca2+ concentration on
thrombin-induced [MC]serotonin secretion, aggregation, and protein phosphorylation were compared in
platelets of SHRSP and WKY rats (Figure 7). Both
platelets released [14C]serotonin to a similar degree
in the absence of Ca2+. However, the enhancement
of thrombin-induced secretion and aggregation by
extracellular Ca2+ was remarkably greater in WKY
rat platelets than in platelets of SHRSP. This difference in enhancement by extracellular Ca2+ was
observed regardless of the type of stimulants, including Ca2+ ionophore A23187 and adenosine diphosphate (data are not shown). At prehypertensive
ages (less than 4 weeks) there was no difference in
the degree of Ca2+ enhancement between platelets
from SHRSP and WKY rats. In contrast to secretion and aggregation, the magnitude of enhancement in malondialdehyde formation by Ca2+ in
platelets of SHRSP was similar to that in WKY rat
platelets at levels below 1 mM Ca2+, and the enhancement became greater in SHRSP than in WKY rats
at levels over 1.5 mM Ca2+.5 Malondialdehyde
formation is regarded as an indicator of phospholipase A2 and cyclo-oxygenase activities in platelets.27
Tomita et al
b)
Defective Platelet Protein Phosphorylation
0)
9
1.0
(mH)
0
1.0
C«>+ lull)
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Thrombin-induced P47 phosphorylation of platelets prelabeled with 32P from SHRSP was not noticeably different from that of WKY rat platelets in the
absence of Ca2+ or in the presence of 1 mM EDTA
(shown in separated points). As was seen in secTetion and aggregation responses, the enhancement in
protein phosphorylation by the presence of extracellular Ca2+ was less in platelets of SHRSP than in
WKY rat platelets. This attenuated enhancement by
Ca2+ in platelets of SHRSP was also demonstrated in
Ca2+ ionophore A23187 stimulation. Thus, the results
obtained here suggest Ca2+ functions are impaired
that are concerned with secretion, aggregation, and
protein phosphorylation, but not those in phospholipase A2, cyclo-oxygenase, or thromboxane A2 synthetase pathways.
Discussion
A marked reduction of aggregation as well as
secretion was observed in platelets of SHRSP compared with those of age-matched WKY rats with the
development of hypertension.1 The degree of abnormality was inversely correlated with the magnitude
of hypertension in various strains of SHR. The
observation of reduced responses to different proaggregators in platelets of SHRSP1 suggested that
the defect appears to be distal to receptor occupation. The hypofunctions of platelets in SHRSP were
revealed not only with washed platelets but also
with platelets prepared by a gel-filtration procedure
with platelet rich plasma and whole blood.28 These
defects are completely different from those found in
platelets from experimentally hypertensive rats23
and were not corrected by hypertensive treatment.3
Platelets of SHRSP have a normal content of
serotonin and adenine nucleotides at less than 20
weeks of age when the rats were killed for the
313
FIGURE 7. Effects of extracelluon
lar Ca2+ concentrations
thrombin-induced ["Cjserotonin
secretion, aggregation, and protein phosphorylation in platelets
from SHRSP and WKY rats.
[l4C]serotonin prelabeled platelets (Panel aj and platelets prelabeled with nP (Panel b and Panel
c)from SHRSP (—•-) and WKY
rats (~o~) were stimulated with
thrombin (0.22 units/ml) for3minutes in Panel a, 0.13 units/ml for
1 minute in Panel b and Panel c
in the presence of 2 mM EGTA
(shown in separated points) and
in varying concentrations ofCa2+
(0, 0.25, and 1.5 mM). Each point
represents mean of triplicates.
SHRSP, stroke-prone spontaneously hypertensive rats; WKY,
Wistar-Kyoto rats.
present experiments. In spite of severe hypoaggregability and reduction of secretion in the presence
of Ca2+, neither malondialdehyde nor thromboxane
B2 formation were affected in platelets of SHRSP5;
they were rather enhanced in platelets of SHRSP
with higher concentration of stimulants. Thrombinstimulated rises in [Ca2+]; in platelets of SHRSP
were slightly delayed, but the maximum [Ca2+]i was
in the same range compared with that of WKY
rats.4 These abnormalities observed in platelets of
SHRSP resembled the defects in human platelets
reported by Hardisty et al.29
Accompanying reduced physiological responses,
thrombin-induced phosphorylation of P47 was markedly decreased in platelets of SHRSP, whereas
TPA-induced aggregation, secretion, and phosphorylation of the protein in SHRSP platelets were
similar to those in WKY rat platelets. Thus, it is
suggested that protein kinase C activity and its
substrate P47 were normally present in SHRSP
platelets, and the defective protein (P47) phosphorylation results from incomplete receptor-mediated
activation of protein kinase C. Reduction of
thrombin-induced P47 phosphorylation in platelets
could possibly result from defects in receptor
functions,1 phospholipase C activity,2 phospholipids of platelet membrane,3 protein kinase C activity,4
P47,5 and others.6 The possibility of receptor malfunction has been excluded in the case of platelets
of SHRSP since hypofunctions were observed with
any stimulants.1
Phosphatidylinositol, PIP, and PIP2 are minor
constituents of mammalian membranes. However,
agonist-induced phosphoinositide breakdown functions as a signal-generating system.6-30 Phosphatidylinositol was 20% less in platelets of SHRSP
while the contents of PIP and PIP2 were not signif-
314
Hypertension
Vol 14, No 3, September 1989
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icantly different between the two strains. In addition, phospholipase C activity is not apparently
related to attenuated responses of aggregation, secretion, and protein phosphorylation in platelets of
SHRSP. Ca2+ dependency experiments of aggregation, secretion, and protein phosphorylation (Figure
5) showed that hypofunctions of platelets of SHRSP
were observed only in the presence of extracellular
Ca2+, or they were exaggerated in such conditions.
Furthermore, Ca2+ ionophore A23187 stimulation
demonstrates more clearly the hypofunctions of
platelets of SHRSP than thrombin stimulation. Since
the abnormalities were observed only in the presence of extracellular Ca2+, the results obtained so
far suggest that Ca2+ function is defective in platelets of SHRSP, first in the receptor-mediated activation of protein kinase C, and second in postprotein kinase C-mediated events coupling to
physiological responses. Recently, it has been demonstrated that P47, a major substrate of protein
kinase C, contains a potential Ca2+-binding "EF
hand" structure and a region that strongly resembles known protein kinase C phosphorylation sites.31
Divergent results on abnormalities of platelet
aggregation and secretion in spontaneously hypertensive rats have been reported. We have consistently observed hypofunctions of platelets from
SHRSP 1 - 52328 - 32 and SHR.23 In addition, these
hypofunctions were demonstrated with any of the
platelet preparations28 (whole blood, platelet rich
plasma, gel-filtered, and washed) and also with
various aggregating agents. 1 The results obtained
by Hamet et al33 coincides well with ours. In
contrast, Koutouzov et al34 and Huzoor-Akbar
and Anwer35 have reported increased aggregation
in platelets of SHR.
We first reported a preliminary result in 198532
that P47 phosphorylation was reduced in platelets
of SHRSP. In this paper the mechanism of the
defective protein phosphorylation related to hypoaggregability was investigated. In 1988, HuzoorAkbar and Anwer33 reported an increase in thrombininduced protein phosphorylation, and Koutouzov et
al34 observed an increase in phospholipase C activity in thrombin-stimulated platelets of SHR. These
results contradict ours that were obtained in platelets of SHRSP. These contradictory results may not
be due to the difference between SHR and SHRSP
because we have shown that hypofunctions of platelets are correlated with blood pressure in the strains
of WKY rats, including SHR, offspring of WKY
rats and SHRSP, and SHRSP. Our findings suggest
that the defect is distal to receptor occupancy,
whereas the observations by Huzoor-Akbar and
Anwer35 and Koutouzov et al34 suggest defective
thrombin receptors. We showed that WKY rats and
another strain of Wistar rat (Wistar Mishima) show
different responses to thrombin, probably due to the
difference in the thrombin receptors.5 Therefore,
these divergent findings on SHR might be ascribed
to biological variability in WKY rats and SHR as
discussed by Kurtz and Morris36 and Yamori.37
In an attempt to find the etiology of hypertension,
it is not important to note the apparent difference
between spontaneously hypertensive rats and control rats, but it is important to demonstrate the
genetic deviation in SHR related to high blood
pressure. Although there are several interesting
reports of in vitro experiments on protein kinase C
in SHR platelets38 and phospholipase C in SHR
aorta,39 our in vitro preliminary results on protein
kinase C suggest that it is extremely difficult to draw
a conclusion for in vivo phenomena from in vitro
experiments, especially in protein kinase C.
Acknowledgment
We are grateful to Prof. Kozo. Okamoto, Department of Pathology, Kinki University Medical School
for pertinent advice.
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KEY WORDS • platelets • protein kinase C • protein
phosphoryiation • genetic hypertension • stroke-prone
spontaneously hypertensive rats
Defective protein phosphorylation associated with hypofunctions in stroke-prone
spontaneously hypertensive rat platelets.
T Tomita, M Ikeda, Y Inoue, T Mitubori and K Umegaki
Hypertension. 1989;14:304-315
doi: 10.1161/01.HYP.14.3.304
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