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Inherited Abnormalities in Platelet Organelles and Platelet Formation and
Associated Altered Expression of Low Molecular Weight Guanosine
Triphosphate-Binding Proteins in the Mouse Pigment Mutant Gunmetal
By Richard T. Swank, Shelley Y. Jiang, Madonna Reddington, James Conway, Dennis Stephenson,
Michael P. McGarry, and Edward K. Novak
Gunmetal(gmlgm)is a recessively inherited mouse pigment
dilution mutant that has high mortality and poor reproductive rates. In these studies, several hematologic defects
were found associated with the mutation, including prolonged bleeding times, together with thrombocytopeniaand
increased platelet size. A unique feature is the presence of
simultaneous abnormalities in two platelet organelles,
dense granules and a-granules. The dense granule component serotonin is present at about half the normal concentration, as are visible dense granules. Three a-granule
components (fibrinogen, platelet factor 4, and von Willebrand factor) are also significantly reduced. Thus, in several
respects the gunmetal mutant resembles the human gray
platelet syndrome. A novel abnormality in expression of low
molecular weight guanosine triphosphate (GTP)-binding
proteins occurs in platelets of gunmetal. In Western blot
assays, two additional GTP-binding proteins of 28.5 and
25 Kd were detected. The abnormal expression of GTPbinding proteins is, like the hematologic defects, genetically
recessive and is tissue specific. Liver, kidney, brain, spleen,
macrophages, and neutrophils have normal GTP-binding
protein expression. The additional GTP-binding proteins are
soluble. The data indicate that platelet formation and
platelet organelle biogenesis are under common genetic
control and that abnormal regulation of GTP-binding proteins may affect one or both processes.
0 1993 by The American Society of Hematology.
M
erogeneous syndrome.” A likely explanation is that this disease is genetically heterogeneous in humans, as has been established by studies in the mouse. Therefore, it will be useful
to have many animal models of human SPD to fully understand and develop therapies for all forms of human SPD.
Inbred mouse strains are a particularly fertile reservoir of
pigment mutants. Mouse fanciers and breeders have, during
the past century, identified more than 60 pigment genesz3
and in most cases have mapped them to specific chromosomal
sites. The mouse gunmetal (gm) recessive mutation arose
spontaneously in the C57BL/6J strain in 1962.24It causes a
grayish-black pigment dilution, although eye color is not affected. Homozygous gunmetal mice have high mortality and
do not breed well.
We provide evidence that the gunmetal mutant has hemorrhagic symptoms together with abnormalities in at least
two platelet subcellular organelles, dense granules and 01granules. In addition, gunmetal has an unusual alteration in
expression of low molecular weight (LMW) guanosine triphosphate (GTP)-binding proteins, recently implicated in the
regulation of the biogenesis and intracellular movement of
OUSE MUTANTS are a rich source of inherited
hemorrhagic abnormalities. Many mutants have
platelet storage pool deficiency (SPD)’-6 and the RIIIS/J
inbred strain has type IA von Willebrand disease.’ Thus far,
no mouse mutant with abnormalities in a-granules has been
described though inherited defects in a-granules have been
described in humans.‘-” The 12 mouse SPD mutants thus
far identified have an accompanying pigment dilution in addition to symptoms typical of SPD, including increased
bleeding times and diminished levels of dense granule components such as serotonin and adenine nucleotides. This suggests that genes that control the biogenesis or processing of
platelet dense granules are similarly operative in melanosome
formation. A comparable association between abnormalities
in platelet dense granules and in melanosomes occurs in the
human Chediak-Higashi ~ y n d r o m e ’ ~and
, ’ ~ HermanskyPudlak ~ y n d r o m e ’ ~and
, ’ ~ in pigment dilution mutants of
other specie^.'^-'^ In many, but not all,3 mouse pigment dilution mutants, there are abnormalities in a third intracellular
organelle, the lysosome.
Recent studies2’.’’ suggest that platelet SPD is, after von
Willebrand disease, the most common cause of prolonged
bleeding in humans. Human SPD is a phenotypically het-
organelle^.^^.'^
MATERIALS AND METHODS
From the Molecular and Cellular Biology Department, and Laboratory Animal Resources, Roswell Park Cancer Institute, Bufalo,
NY;and the Hematology Laboratory, Buffalo General Hospital, Buffalo, NY.
Submitted September 17, 1992; accepted December 23, 1992.
Supported by Grant No. HL 31698from the National Heart, Lung
and Blood Institute.
Address reprint requests to Richard T. Swank, PhD. Dept. ofMolecular and Cellular Biology, Roswell Park Cancer Institute, Carlton
and Elm Sts, Buflalo, NY 14263.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I993 by The American Society of Hematology.
0006-4971/93/81I0-0021$3.00/0
2626
Animals. Gunmetal (gm/gm) mice, maintained on the B6C3Fe
stock background, and other mutant mice were originally obtained
from the Jackson Laboratory (Bar Harbor, ME) and subsequently
bred at the animal care facilitiesof the Roswell Park Cancer Institute.
The gunmetal mutants were maintained by breeding heterozygous
gm/+ mice by homozygous gm/gm mice.
Bleeding times. Bleeding time was determined by tail bleeding
as des~ribed.~.~’
Platelet collections and counts. Platelets were collected for serotonin determinations, thrombin-stimulated secretion studies, and
Western blotting as described.’* Platelets were counted (3,00O/analysis) and sized in citrated whole blood in a Coulter Model S f - 4
analyzer (Hialeah, FL) with channels set so that all particles between
2.0 and 20 fim3were counted as platelets.
Platelet serotonin assay. Platelets were lysed in 1 mL distilled
water and assayed fluonmetrically for serotonin according to Crosti
and L ~ c c h e l l i . ~ ~
Blood. Vol81, No 10 (May 15).1993:pp 2626-2635
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ABNORMAL PLATELETS AND GTP BINDING IN GUNMETAL
Electron microscopy. Platelet-rich plasma was collected from citrated, undiluted blood by centrifugation at l5Og for 10 minutes.
Unfixed and unstained platelets were rapidly airdried on carboncoated grids.30The number of dense granules larger than 50 nm was
counted in individual platelets as d e ~ r i b e d . ~
Thrombin-stimulated platelet secretion. To platelet-rich plasma,
0.5 pCi I4C serotonin (Amersham Corp, Arlington Heights, IL) was
added and the mixture was allowed to incubate for 30 minutes at
37°C. Platelets were washed twice with phosphate-buffered saline,
pH 7.4, containing 2% bovine serum albumin. Platelets were resuspended at 1 X 108/mLin 1.0 mL Tyrode’s solution (minus calcium
plus magnesium). Thrombin, at various concentrations in 0.1 mL,
and 0.85% NaCl were added and incubation was continued at 37°C
for 3 minutes with constant shaking. The reaction was stopped with
2.5 nmol/mL Thromstop (American Diagnostics Inc, Greenwich,
CT), a synthetic competitive inhibitor of thrombin,” and tubes were
immersed in ice. Platelets were pelleted and then resuspended in 1.O
mL Tyrode’s solution. The pellet and supernatant were made 1% in
Triton X-LOO. Aliquots were assayed for radioactivity and lysosomal
enzyme activity. @-Glucuronidaseand P-galactosidase were assayed
with fluorescent methylumbelliferyl s~bstrates.’~
Lysosomal enzyme concentrations in kidney and liver extracts of
testosterone-treated female mice were assayed as described:
Western blot assays of platelet factor 4 (PF4)fibrinogen, von Willebrandfactor (v WF), and ras-relatedproteins. Extracts of platelets
( I O to 60 pg protein) were electrophoresed on denaturing sodium
dodecyl sulfate (SDS) acrylamide gels under reducing conditions as
described.” In both these assays and the detection of GTP-binding
proteins described below, a proteinase inhibitor cocktail consisting
of (final concentration) 1 mmol/L EDTA, 1 pg/mL chymostatin, 5
pg/mL aprotinin, 5 pmol/L leupeptin, 5 pmol/L pepstatin, and 1
mmol/L phenylmethylsulfonyl fluoride (PMSF) was included. The
proteinase inhibitors were added during platelet collection and subsequently maintained in all platelet extract solutions. PF4 was quantitated on Western blots using a rabbit polyclonal antibody to rat
PF434at 200: 1 dilution, followed by ‘251-labeledgoat antirabbit secondary antibody (ICN Biomedicals, Inc, Irvine, CA) at 1,000:l dilution (0.4 pCi/mL). Radioactivity in PF4 components was determined, at two quantities of platelet protein that differed by twofold,
by storage phosphor technology with a Molecular Dynamics
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The storage
phosphor screens store energy transferred from radioactive bands of
Western blots. The exposed phosphor screens are, in turn, read in
the PhosphorImager, where they are scanned with a focused HeNe
laser to release the stored energy. The blue light emitted is collected
and measured for each pixel. In comparison to traditional x-ray film
autoradiography, storage phosphor screen autoradiography is faster
and quantitatively linear over a wider dynamic range. Radioactivity
in specific PF4 bands was corrected for background radioactivity in
a region of the Western blot having no visible labeled bands. In some
cases, a peroxidase-coupled goat antirabbit secondary antibody (Kirkegaard and Perry, Gaithersburg MD) was used at 1,OOO: 1 to increase
the sensitivity of detection of PF4.
In a similar fashion, fibrinogen and vWF were assayed in normal
and mutant platelets. Rabbit antiserum to human fibrinogen was
from Diagnostica Stago (Asnieres, France) and was used at 500:l
dilution in Western blots. Rabbit antiserum to human factor VI11
(FVII1)-related antigen was from Accurate Chemical and Scientific
Corp (Westbury, NY) and was used at 500: 1 dilution.
Western blotting procedures were also performed using antibodies
to human C-H ras (Oncogene Science, Inc, Manhasset, NY) to rat
V-H ras (a gift of Dr G. Anderson, Roswell Park Cancer Institute,
Buffalo, NY) and to peptide 57-76 of rat rab-4 (a gift of Dr J. Lenhard,
Washington University, St Louis, MO).
2627
Plasma vWF assay. Plasma vWF was assayed by a sandwich
enzyme-linked immunosorbent assay (ELISA) procedure using 1:
500 rabbit antihuman vWF (Dako Corp, Carpinteria, CA) Ig fraction
as the coating antibody and 1:4,000 peroxidase-conjugated anti-vWF
(Dako Corp) as the detecting antibody, according to manufacturer’s
instructions. ELISA reaction products were quantitated at 405 nm
on a Bio-Tek EL309 microplate autoreader (Bio-Tek Instruments,
Highland Park, VT).
Detection of GTP-bindingproteins. Extracts of platelets and other
tissues (30 to 70 pg protein) in the above described proteinase inhibitor
solutions were electrophoresedon 12%denaturing SDS gels and blotted to nitrocellulose essentially as described by Lapetina and R e e ~ . ~ ’
Use of freshly prepared mercaptoethanol in the SDS solution used
to denature the platelet proteins, before the first electrophoresis, reduced variability in resolution of GTP-binding proteins. A modification was that 20% methanol and 0.1% SDS was included in the
transfer buffer and [’*P]GTP was increased to 2 pCi/mL during incubation of Western blots. Binding of GTP to specific platelet proteins
was detected by incubation of blots with [cY-~’P]GTP
followed by
autoradiography.
To assay GTP-binding proteins in membrane and soluble fractions,
platelets at 0.3 X 108/mL in hypotonic buffer ( 5 mmol/L Tris-HC1,
5 mmol/L EDTA, pH 7.5) were sonicated for 10 seconds at setting
1.5 in a Heat Systems Ultrasonic Processor W-220 (Heat Systems
Ultrasonics, Inc, Farmingdale, NY) at 0°C. Sonicated extracts were
centrifuged at 1,000g for 10 minutes to remove unbroken cells and
debris. The supernatant was then centrifuged at 100,OOOgfor 1 hour.
This pellet was resuspended in the original volume of starting buffer
and taken as the membrane fraction, and the supernatant was used
as the soluble fraction. Extracts of whole platelets, membrane, and
soluble fractions derived from an equivalent number of platelets were
electrophoresed, blotted, and incubated with [32P]GTP.
RESULTS
Abnormal hemostasis in gunmetal mice. Prolonged
bleeding times were found in all gm/gm mice tested (Table
1). In 11 gm/gm mice, bleeding times were greater than 15
minutes; in one other, bleeding time was prolonged to 8 minutes. In contrast, bleeding times in all seven gm/+ mice were
normal (less than 5 minutes), thus establishing the recessive
nature of the bleeding abnormality. Bleeding times were normal in a second recessive pigment dilution mutant, slaty
.36
When platelet number and size were determined (Table
l), it was apparent that gm/gm mice have both thrombocytopenia and somewhat increased platelet size. Platelet counts
of gm/gm mice were approximately 50% those of gm/+ and
mean platelet volumes of gm/gm were 30% larger. It is likely
the mean platelet volume of 6.7 fL in gunmetal represents a
minimum estimate because of the difficulty of distinguishing
large platelets from small erythrocytes in whole blood analyses. This platelet size difference was maintained in gunmetal
mice over a wide age span (from 4 weeks to 6 months).
Further analyses of peripheral blood of mutant mice
showed no other significant quantitative abnormalities for
total white or red blood cell numbers or hemoglobin concentrations in gunmetal. Also, microscopic analysis of blood
smears stained with Wright’s stain showed no significant
qualitative abnormalities of blood cells in the gm/gm mutant
as compared with gm/+. Dohle bodies, similar to those characteristic of the May-Hegglin anomaly, were not observed in
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SWANK ET AL
2628
Table 1. Prolonged Bleeding Time and Platelet Abnormalities in Gunmetal Mice
Genotype
gml+
gmlsm
Shf+
sltislt
Bleeding Time
(mid
*
1.4 0.09(7)
> I 5 (1 1)’
8.0(1)
2.4 k 0.48(4)
2.3 t 0.2214)
Platelet Count
(XlOJ/flL)
913 f 50 (9)
466 ? 36 (6)’
Mean Platelet
Volume (fL)
Serotonin ( + g / l O g )
Platelets
Dense Granules/
Platelet
5.1 k0.12 (9)
6.7f 0.32(9)t
3.02f 0.23(7)
1.43f 0.23 (7)t
4.59 % 0.52(61)z
9.52k 0.90(50)
Values represent the mean f SEM of the number of mice in parentheses (except for dense granules/platelet, in which the values in parentheses
are the number of individual platelets analyzed). Values for +/+ mice are not included, as no significant differences were noted between gm/+ and
+/t mice in the above measurements.
P I .001.
t P s .01.
t P I ,0002.
4
peripheral blood granulocytes of gm/gm mice (Dr Judith
Wall, personal communication, 1992).
Dense granule abnormalities. By two criteria, gm/gm
mice exhibit a mild SPD (Table I). First, the concentration
of serotonin, a typical dense granule constituent, is reduced
to less than half of the control values. Second, the number
of dense granules per platelet detectable after whole mount
electron microscopy is similarly reduced to about 50% of
control values. A comparative distribution of morphologically
observable dense granules in platelets of gm/gm and gmlt’
is given in Fig 1. In gm/+ platelets, the mean number of
dense granules per platelet is 9 to 10, with rare platelets having
as few as 0 or as many as 30 granules per platelet. In contrast,
the distribution in gm/gm is skewed toward fewer granules
per platelet. In fact, 14 of 61 platelets had no observable
dense granules. Because gunmetal platelets are, on average,
larger than normal platelets (Table I), it follows that the dense
granule deficiency, per platelet volume, is larger than that
indicated in Fig 1. Because of the larger volume of gunmetal
platelets, the measured dense granule deficiencies are minimal
estimates.
a-Granule abnormalities. Western blotting procedures
were used to enumerate platelet a-alpha granule proteins.
Specific immunoradiolabeled components were quantitated
on blots by storage phosphor technology. Platelet fibrinogen
was detected as a set of three bands between 57 and 66 Kd
(Fig 2A). The intensity ofthese radiolabeled components was
equivalent in -t/+and gm/+ platelets and was reduced in
gm/gnz platelets, indicating that the reduction of fibrinogen,
like the pigment dilution in gunmetal, is genetically recessive.
An additional band of unknown origin at 38 Kd was present
at equal intensity in all samples.
Similar results were obtained in analyses of platelet vWF
(Fig 2B). A major band at 240 Kd was present at equal quantities in +/+ and gm/+ platelets and was reduced in gm/gm
platelets.
Immunoblots for PF4 showed three components of
expected34LMW. Two minor bands appeared at 16 and 15
Kd and a major form appeared at 13 Kd (Fig 2C). Platelets
from mice homozygous for gunmetal showed a considerable
reduction specifically in these LMW bands. The intensities
of these bands were similar in gm/+ and +/+ mice. There
were also several additional bands of unknown origin at higher
molecular weight that were present at equal intensity in mice
of all three genotypes. These are likely nonspecific components that appeared because of the low cross-reactivity of the
antibody to rat PF4 with mouse PF4 and the subsequent
necessity of using ultrasensitive detection procedures. Because
the nonspecific components were sufficiently removed From
the specific LMW PF4 bands, they did not interfere with the
analyses.
When specific fibrinogen, vWF, and PF4 components were
quantitated by phosphor storage technology (Table 2), a general reduction of all three components to 40% to 50% of
I
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
22
30
Dense Granules Per Platelet
14
13
--
is”=
B
0 I 2 3 4 5 6 7 8 9 10 I 1 12 13 14 15 16 17 18 19
Dense Granules Per Platelet
Fig 1. Distributionsof dense granules in platelets from (A)normal
@m/ ) and (B) gunmetal @m/gm)mice. Platelets were air-dried,
and unstained whole platelets were examined for dense granules
by electron microscopy. The number of platelets analyzed was 50
@m/ ) and 61 @m/gm)from three mice each. Values for ( / 1
platelets are not included as no significant differences were noted
between gm/ and 1 .
+
++
+
+
++
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ABNORMAL PLATELETS AND GTP BINDING IN GUNMETAL
2629
20097.46946 -
3021.5-
200 Fig 2. Reduced concentrations of @-granule
components in plateletsof gunmetal. Equalamounts
of protein from platelet extracts of three separate
1 , gm/ ,and gmlgm mice were electrophoresed and Western blotted. Nitrocellulose membranes were treated with antibody to (A) fibrinogen,
(B) vWF, or (C) PF4. In the fibrinogen and vWF analyses, this was followed by treatment with [1261]labeled secondary antibody and autoradiography. In
PF4 analyses, peroxidase-labeledsecondary antibody was used for increased sensitivity. The PF4
components are indicated by a bracket.
+ +
+
97.469 -
46 -
normal levels was observed. Also, in all three cases, the reduction was genetically recessive, ie, mice heterozygous for
the gunmetal gene had normal concentrations.
Plasma of gunmetal mice was analyzed to determine if
there was an increase in plasma vWF concomitant with the
platelet vWF reduction. Indeed, gm/gm plasma vWF units
(154 k IO U/dL) were significantly(P< .02) increased relative
to gm/+ plasma units (9 I k 12 U/dL).
Normal 1.vsosomal enzyme Concentrations and thrombinstimulated secretion. No abnormalities were detected in
another platelet subcellular organelle, the lysosome. Concentrations of two lysosomal enzymes, B-glucuronidase and pgalactosidase, were normal in gm/gm mice (Table 3).
In the case of several other mouse pigment mutants with
SPD,'*2.4.s
there is an associated increase in concentrations
of kidney lysosomal enzymes due to a lowered rate of secretion of kidney lysosomal enzymes into urine. However, kidney glucuronidase and galactosidase concentrations were
normal in gm/gm mice, as were concentrations of these two
enzymes in a control tissue, liver (Table 3). Likewise, measurements of thrombin-stimulated secretion from platelets
of the dense granule component serotonin and two lysosomal
enzymes showed only slight decreases in the secretion
of serotonin and glucuronidase from gunmetal platelets
(Table 4).
Altered expression of GTP-binding proteins in gunmetal
platelets. Several LMW (27.5,26, and 23 Kd) GTP-binding
proteins were detected (Fig 3) in normal (+/+) platelets when
Western blots of platelet extracts were probed with ["PJGTP.
The pattern of expression in homozygous gunmetal (gm/gm)
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SWANK ET AL
2630
46 '
I
30 -
was quite different in that at least two additional LMW GTPbinding proteins were apparent, one of higher molecular
weight (28.5 Kd) and another of 25 Kd. In addition, all GTPbinding proteins expressed in normal platelets were expressed
in gunmetal platelets. The degree of resolution of the normal
23-Kd GTP-binding proteins was variable in both normal
and mutant mice. Recent experiments have shown that the
degree of resolution of GTP-binding proteins is sensitive to
the reducing environment during SDS electrophoresis. Use
of freshly prepared mercaptoethanol and/or dithiothreitol
reduces this variability. The 28.5- and 25-Kd GTP-binding
proteins were consistently observed in platelets from 14 separate gm/gm mice and were never observed in platelets from
12 gm/+ mice. No difference in expression of platelet LMW
GTP-binding proteins was observed between male and female
mice.
The proteins detected by this assay are GTP specific. Inclusion of IO pmol/L GTP or GTPyS in the [32P]GTPincubation buffer resulted in no detectable radiolabeled bands
in either +/+ or gm/gm platelet extracts, an effect not observed with I O pmol/L adenosine triphosphate (ATP) or guanosine monophosphate (GMP) (not shown). In fact, increased
[32P]GTP-bindingto all proteins was observed in the presence
of ATP or GMP.
The total amount of LMW GTP-binding proteins in gunmetal platelets, as detected by densitometric scans of Western
blots, is increased 2. I-fold over that of normal platelets. The
increased expression in gunmetal platelets is entirely due to
expression of the new 28.5- and 25-Kd GTP-binding proteins.
Expression of the remaining GTP-binding proteins is equal
in normal and gunmetal platelets.
The expression of the additional LMW GTP-binding proteins is genetically recessive. The pattern in platelets from
heterozygous (gm/+) mice is identical to that of normal (+/
+) platelet, (Fig 3). This result is in agreement with the recessive nature of other characteristics in gm/gm mice.
The abnormal expression of gunmetal GTP-binding proteins is specific to platelets. Western blots of extracts from
brain, kidney, spleen, and bone marrow (Fig 4) showed tissue-
Table 2. Reduction of a-Granule Components in gm/gm Platelets
Table 3. Concentrations of Lysosomal Enzymes in Kidney, Liver.
and Platelets of gm/+ Normal and gm/gm Mutant Mice
Relative Units
+/+
gm/+
"9
Fibrinogen
vWF
PF4
664 f 43
647 f 32
266 f 31'
376 f 21
447 f 34
206 f 19'
348 f 33
343 f 32
162f 17t
Platelet extracts (50 pg protein) were electrophoresed, transferred to
nitrocellulose membranes by Western blotting, and treated with antibodies to either fibrinogen, vWF, or PF4 followed by ['261]-labeledsecond
antibody. Relative units of radioactivity in specific fibrinogen, vWF, or
PF4 bands were determined by phosphor storage technology. Results
are expressed as the mean f SEM of determinations on extracts from
three mice of each genotype.
P s .01.
t P s .02.
~~
Tissue
Kidney
Liver
Platelets
Genotype
gm/+
"g
P I +
gmlgm
gm/+
gmlgm
@-Glucuronidase
8-Galactosidase
1 1 8 2 17
112 f 6.7
22.0 f 0.67
22.4 f 1.1
0.0437 f 0.0028 (8)
0.0423 f 0.0040 (7)
25.9 f 1.7
18.0 f 2.7
9.58 f 1.0
10.1 f 0.39
0.0745 2 0.0023 (8)
0.0656 f 0.0050 (7)
Kidney and liver tissues were from mice treated with testosterone as
described in Materials and Methods. Plateletswere from untreated male
or female mice. Values are the mean f SEM of the number of mice
indicated in parentheses. Values for kidney and liver are units per gram
of tissue. Values for platelets are units per milligram of protein.
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ABNORMAL PLATELETS AND GTP BINDING IN GUNMETAL
263 1
Table 4. Thrombin-Stimulated Secretion of Platelet Dense
Granule Contents and Lvsosomal Enzvmes
Thrombin 1UI
0
0.05
0.25
8.7 f 2.4
11 f 1.8
33 f 1.7
26 f 8.2
80 f 3.0
57 f 3.8'
8.3 f 3.8
9.3 f 2.2
8 f 3.8
9.3 f 2.2
20 f 0.33
15 f 1.5*
11 f 4.4
12 f 2.0
25 f 4.2
21 f 1.5
2.5
'*C-serotonin
s"+
wlgm
8 8 " 1.7
72 f 0.9t
@-Glucuronidase
gml+
"9
@-Galactosidase
5.7 f 2.2
gml+
4.6 f 0.88
gmlm
29.0 f 2.5
23.3 f 1.5
36 f 3.2
30 f 3.8
Total cpm ['Tjserotonin initially incorporated in gm/+ and gmlgm
platelets were 10,700 and 10,400, respectively, per lo8 platelets.Values
are the mean percentagesecretion f SEM of measurementson platelets
from three experiments with three mice in each experiment.
* P s ,001.
tPs.01.
P s .05.
*
in platelet formation are suggested by the lowered platelet
count and an accompanying mild increase in platelet volume.
It is possible that the increase in platelet volume is a result
of accelerated platelet production in response to the thrombocytopenia. Associated with this macrothrombocytopenia
are abnormalities in at least two organelles. Dense granules
and their serotonin content are approximately halved and
the a-granule components fibrinogen, vWF, and PF4 are at
40% to 50% of normal levels. Gunmetal is the first mutant
mouse model reported to exhibit both dense granule and agranule abnormalities.
The fact that several a-granule components are reduced
in platelets of gunmetal indicates a general effect on organelle
biogenesis or processing rather than on specific a-granule
components. The finding that vWF that is absent in platelets
is present as excess plasma vWF suggests an abnormality in
targeting or retention of a-granule components rather than
a defect in synthesis. No morphologic abnormalities of gunmetal platelets have been noted at the electron microscope
specific patterns of expression. However, each tissue pattern
was equivalent in gunmetal and normal extracts. In other
experiments (not shown), equivalent GTP-binding protein
expression was observed in extracts from normal and gunmetal liver, neutrophils, macrophages, and retinal pigment
epithelium.
Because platelet SPD was found in gunmetal, platelet extracts of other pigment mutant mice previously shown to
have SPD'*5were tested for abnormal expression of GTPbinding proteins (Fig 5). A normal GTP-binding protein pattern was apparent in the pallid, pale ear, muted, ruby-eye-2,
pearl, light ear, and beige mutants. In other experiments (not
shown), normal GTP-binding protein expression was also
observed in platelet extracts of other mouse pigment mutants
with platelet SPD, including the cocoa, mocha, muted, sandy,
and ruby-eye mutants.
A hallmark of many LMW GTP-binding proteins is that
they are located at specific subcellular membrane site^.'^.*^
In fact, the GTP-binding proteins of normal @I/+) platelets
are predominantly located in the membrane fraction (Fig 6).
The 28.5- and 25-Kd GTP-binding proteins specifically expressed in gunmetal platelets are, on the other hand, concentrated in the soluble fraction (Fig 6). In somc cases, the
subcellular locations of GTP-binding proteins change in response to specific external signals." However, treatment of
gunmetal or normal platelets with the potent agonist thrombin (2.5 U/mL for 3 minutes) did not cause a shift of the
abnormal GTP-binding proteins of gunmetal to the membrane fraction, nor in fact did it affect the subcellular distribution of any GTP-binding protein of either gunmetal or
normal platelets (not shown).
DISCUSSION
The hemorrhagic abnormalities of the gunmetal mutant
are unusual in that they include not only prolonged bleeding
but also apparent widespread abnormalities in both platelet
formation and in platelet organelle biogenesis. Abnormalities
\
$
%
\
%
- 200
- 97
- 69
- 46
- 30
- 21.5
- 14.3
Fig 3. Abnormal expression of LMW GTP-binding proteins in
platelets of gunmetal mice. Platelet extracts from gmlgm. gm/ ,
and / mice, equivalent in protein, were electrophoresedon SDS
gels and Western-blotted to nitrocellulose membranes that were
incubated with [32P]GTPfollowed by autoradiography. The a m w s
indicate the 28.5- and 25-Kd GTP-binding proteins specific to gml
gm platelets.
++
+
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SWANK ET AL
2632
$ / %
\ "$1\
$1
\
$1
81%
@\"
\
\
\
$
1
$/ $/ $/
200.
97.4
69.
46
'
30,
21.5
14.3
J - I +L+J+
Brain
Kidney
Spleen
Bone
Marrow
Fig 4. LMW GTP-bindingproteinsare similarlyexpressed in other
organs of gunmetal and normal mice. Extracts of brain, kidney,
mice equivalent in
spleen, and bone marrow of gmlgm and gm/
protein were analyzed for GTP-binding proteins as described in
Fig 3.
+
level other than a reduced number of a-granules (R.T.S.,
unpublished observations) and the described reduction in
dense granule number.
Gunmetal has some similarities to several inherited human
hemorrhagic diseases. It is similar to Hermansky-Pudlak
syndrome (HPS)I3.l4in that both have pigment dilution and
SPD. However, they differ in that the SPD is more severe in
HPS, and macrothrombocytopenia and a-granule abnormalities are not symptomatic in HPS. There are also similarities of the gunmetal phenotype to human a6SPD' in that
both have deficiencies of a-granules and dense granules.
However, these human patients have a more severe deficiency
of dense granules as well as more variable deficiencies of agranule components. The human May-Hegglin anomaly3'
and Bernard-Soulier syndrome39patients have, like gunmetal,
large mean platelet volumes. May-Hegglin differs from gunmetal in that it exhibits leukopenia and Dohle bodies in
granulocytes and has an autosomal dominant mode of inheritance. The platelet enlargement in Bernard-Soulier patients is considerably greater than that in gunmetal, and the
number of a-granules in Bernard-Soulier syndrome is normal
or often increased.
Among inherited human bleeding diseases, gunmetal is
most similar to the human gray platelet syndrome9-" in that
both have deficiencies of platelet a-granule proteins, reduced
platelet numbers, and enlarged platelet volumes. The a p
pearance of platelet a-granule proteins in plasma is likewise
common to both. Gray platelet patients usually have a more
marked deficiency of a-granule components and do not ex-
hibit SPD. However, the SPD difference may be more a p
parent than real because SPD in gunmetal is mild (twofold
decrease in dense granule contents) and because a similar
twofold variation in platelet dense granule contents is found
among human controls.22
In several respects, the hemostatic abnormalities of gunmetal resemble those recently reported for the Wistar-Furth
rat.4042Both have macrothrombocytopenia accompanied by
partial deficiencies in a-granule components that are inherited
recessively. Another interesting similarity is that both have
lowered reproductive rates. A difference is that the size of
gunmetal platelets does not increase with age as has been
found for the Wistar-Furth rat.
Gunmetal represents the twelfth mouse mutant with combined pigment dilution, prolonged bleeding, and platelet SPD
symptoms.'-6 Thus far, 13 of 36 pigment dilution mutants
surveyed have SPD. However, the abnormalities in hemostasis in gunmetal are quite distinct and more widespread in
nature as compared with the other mouse pigment mutants.
It is the only one with either decreased platelet number or
increased platelet volume. The SPD is milder in gunmetal.
In all other mouse SPD mutants, the highest concentrations
of dense granule components or of morphologically detectable
dense -nranules are 20% of normal or less, whereas dense
granule components in gunmetal platelets are 50% of normal.
Another difference is that deficiencies of a-granule components have not been detected in other mouse mutants with
SPD (R.T.S., unpublished observations). Finally, gunmetal
is the only mouse hemorrhagic mutant with abnormal
expression of LMW GTP-binding proteins.
LMW GTP-binding proteins comprise a very large multigene superfamily, including the ras, rho, rub, ran, and atf
~ubfamilies.4~
They have been implicated in the regulation
of intracellular vesicular traffic and function.25*26.""8Individual GTP-binding proteins are often localized to specific
subcellular organelles and are thought to impart specificity
to the membrane budding and fusion reactions involved in
intracellular vesicle traffic, thereby ensuring accurate delivery
of transport vesicles to their correct targets. Although the
biochemical and molecular properties of LMW GTP-binding
proteins have been well studied, their organellar function(s),
in mammalian systems and particularly in platelet^:^ are
little understood.
The altered expression of GTP-binding proteins in gunmetal platelets is not likely due to simple redistribution of
preexisting normal GTP-binding proteins because "normal"
GTP-binding proteins are present in gunmetal platelets at
the same quantity as they are found in normal platelets. It
appears that new GTP-binding proteins of 28.5 and 25 Kd
are synthesized or otherwise expressed in gunmetal platelets.
The normal allele at the gunmetal gene serves as a dominant
suppressor of expression of the GTP-binding proteins because
no abnormal expression of GTP-binding proteins is apparent
in heterozygous mice. However, additional studies are required to determine if the product of the gunmetal gene directly regulates expression of GTP-binding proteins.
The identities of the new 28.5- and 25-Kd GTP-binding
proteins expressed in gunmetal platelets are unknown. The
possibilities are large because in platelets alone at least 15
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ABNORMAL PLATELETS AND GTP BINDING IN GUNMETAL
2633
200 -k
97-
'
46 -
30Fig 5. The abnormal expression of LMW GTPbinding proteins is specific to gunmetal among
mouse pigment mutants. Platelet extracts, equivalent in protein from normal C57BL/6J and various
pigment mutant mice with SPD, were analyzed for
GTP-binding proteins as described in Fig 3.
22 -:
.d
14 -.
LMW GTP-binding proteins have been identified."9 The 28.5and 25-Kd GTP-binding proteins do not react to antibodies
to C-H rus or V-H rus or rab-4 GTP-binding proteins in
Western blots (R.T.S., unpublished observations).
The abnormal expression of GTP-binding proteins exhibits
a striking tissue specificity. Among nine tissues tested, it was
found only in platelets. The normal expression patterns in
bone marrow and spleen are not indicative of normal expression in megakaryocyte precursors of platelets because abnormal GTP-binding proteins expression in a minor cell
population would not be detected by these techniques. The
normal expression in retinal pigment epithelium is consistent
with normal eye color in gunmetal mutants.24 It will be interesting to determine if, reflective of the coat color dilution,
GTP-binding protein expression is altered in skin melanocytes
of gunmetal given the gene's effect on coat color.
Because it has been established that LMW GTP-binding
proteins are involved in organellar formation and function
in other system^:^.^^ it may be speculated that the abnormal
expression of LMW GTP-binding proteins cause the dense
granule and a-granule abnormalities in gunmetal mice. An
abnormality in LMW GTP-binding proteins expression in
Fig 6. The LMW GTP-binding proteins that are
abnormally expressed in gunmetal platelets are
concentrated in the soluble subcellular fraction.
Membrane and solublefractions were preparedfrom
normal and gunmetal platelets and analyzed, together with an amount of total platelet extract
equivalent to that from which the subcellular fractions were prepared, for GTP-binding proteins as
described in Fig 3.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2634
SWANK ET AL
megakaryocytes might result in abnormal biogenesis of
platelet granules. However, these experiments establish that
grossly abnormal expression of platelet LMW GTP-binding
proteins is not obligatorily associated with SPD because 11
other mouse mutants with SPD have no obvious abnormalities in these proteins.
The abnormal GTP-binding proteins in gunmetal are not
involved in the regulation of secretion of platelet organelles
because there is little or no effects on secretion of dense granules or lysosomes in gunmetal. Also, the fact that the subcellular location of the abnormal GTP-binding proteins does
not change in response to thrombin treatment is consistent
with the notion that they do not regulate secretion. Physiologic
stimuli cause changes of the subcellular location of GTPbinding proteins of the secretory pathway in other systems.37,50,5I
An alternative possibility is that the abnormal expression
of GTP-binding proteins in gunmetal causes the abnormal
platelet formation. Platelet formation from megakaryocytes
includes extensive membrane budding and fusion.’* Therefore, it is reasonable to speculate that GTP-binding proteins
are involved in platelet formation because in general they
regulate membrane budding and f ~ s i o n The
. ~ ~fact
, ~ that
~ the
gunmetal GTP-binding protein abnormality is found only in
platelets is consistent with a defect in a platelet-specific process, such as platelet formation. Also, this interpretation is
consistent with the fact that, among all the 12 mouse mutants
with platelet organellar abnormalities, only the gunmetal
mutant, with its unique abnormalities in GTP-binding proteins, has an accompanying abnormality in platelet formation.
It may be relevant that the GTP-binding protein CDc4253
is involved in regulation of cell polarity and budding in yeast.
Establishment ofcell polarity in yeast appears to be mediated
by the actin cyt~skeleton.~~
Evidence for a role of the megakaryocyte cytoskeleton in platelet formation has been preof GTP-binding protein regulation in
~ e n t e d .Analyses
~~
gunmetal megakaryocytes will contribute to the resolution
of this proposal.
The combined abnormalities in gunmetal suggest that formation of platelets and their organelles may be under common genetic control. The gunmetal mouse provides a model
of hereditary macrothrombocytopenia that should be useful
in studying the determination and regulation of platelet formation and/or organelle biogenesis during megakaryocyte
differentiation and the role of GTP-binding proteins in these
processes.
ACKNOWLEDGMENT
We thank Dr David J. Kuter of the Department of Biology of the
Massachusetts Institute of Technology for the gift of rabbit antiserum
to rat platelet factor 4. Ed Hurley and A1 Cairo analyzed platelets by
electron microscopy, whde John Mierzwa and Roger Palmer provided
platelet counting and sizing. We thank Jie Wang for able technical
assistance and Cheryl Mrowczynski and Cynthia Bates for expert
secretarial assistance.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1993 81: 2626-2635
Inherited abnormalities in platelet organelles and platelet formation
and associated altered expression of low molecular weight guanosine
triphosphate-binding proteins in the mouse pigment mutant gunmetal
RT Swank, SY Jiang, M Reddington, J Conway, D Stephenson, MP McGarry and EK Novak
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