Platelet Storage Pool Deficiency Associated With Inherited Abnormalities of the Inner Ear in the Mouse Pigment Mutants Muted and Mocha

By Richard T. Swank, Madonna Reddington, Orla Howlett, and Edward K. Novak

Several inherited human syndromes have combined platelet, auditory, and/or pigment abnormalities. In the mouse the pallid pigment mutant has abnormalities of the otoliths of the inner ear together with a bleeding abnormality caused by platelet storage pool deficiency (SPD). To determine if this association is common, two other mouse pigment mutants, muted and mocha, which are known to have inner ear abnormalities, were examined for hematologic abnormali- ties. Both mutants had prolonged bleeding times accompa- nied by abnormalities of dense granules as determined by whole mount electron microscopy of platelets and by label- ing platelets with mepacrine. When mutant platelets were treated with collagen, there was minimal secretion of adeno- sine triphosphate and aggregation was reduced. Lysosomal

UTED IS AN AUTOSOMAL recessive pigment M gene on mouse chromosome 13 that causes light eyes at birth and fur of a muted brown shade.’ The gene causes changes in postural reflexes; the head is often tilted to one side. These abnormalities are associated with an absence or deficiency of otoliths of the inner ear.

Mocha is an autosomal recessive pigment mutant on mouse chromosome 10.’ It has an absence of visible pigment in the eyes, which darken to a deep red in adults, and pigment of the coat is diluted. Unusual postural reflexes are present and are likewise accompanied by abnormalities of the otoliths and other organs of the inner ear.

In their effects on pigmentation and the inner ear, the muted and mocha mutants resemble the mouse autosomal recessive pigment mutant pallid? The defect in the pallid mutant often causes the head to be tilted to one side, and the mice may be unable to orient themselves if submerged in water. Most pallid mice have no otoliths of the inner ear.4,5 Other studies6-” have shown that the pallid mutant has abnormalities of several intracellular organelles includ- ing melanosomes, lysosomes, and platelet dense granules, and that the platelet defect is accompanied by a bleeding abnormality.

In humans, a similar triad of abnormalities in platelet dense granules, lysosomes, and in pigmentation occurs in the Chediak-Higashi syndrome (CHS)’’,’’ and the Herman- sky-Pudlak syndrome (HPS).”-13 The abnormality of plate-

From the Department of Molecular and Cell Biology, Roswell Park

Submitted Februay 19, 1991; accepted June 20, 1991. Supported by Grant No. HL 31698from the National Heart Lung

and Blood Institute. Address reprint requests to Richard T. Swank, PhD, Department of

Molecular and Cellular Biology, Roswell Park Cancer Institute, Carlton and Elm Sts, Buffalo, Ny14263.

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 1734 solely to indicate this fact.

Cancer Institute, Buffalo, Ny:

0 1991 by The American Society of Hematology. 0006-4971 I91 17808-0013$3.00/ 0

enzyme secretion in response to thrombin treatment was partially reduced in muted platelets and markedly reduced in mocha platelets. Similar reductions in constitutive lysosomal enzyme secretion from kidney proximal tubule cells were noted in the two mutants. These studies show that several mutations that cause pigment dilution and platelet SPD are associated with abnormalities of the inner ear. Also, these mutants, like previously described mouse pigment mutants, are models for human Hermansky-Pudlak syndrome and provide additional examples of single genes that simulta- neously affect melanosomes, lysosomes, and platelet dense granules. 0 1991 by The American Society of Hematology.

let dense granules is manifest as platelet storage pool deficiency (SPD), which is now recognized’e16 as a relatively common and heterogeneous inherited syndrome.

Auditory abnormalities have been described in CHS and HPS and in other inherited hypopigmentation conditions including classical albinism18 and Waardenburg’s syndrome.” Therefore, it is of interest to determine if there are similar associations of bleeding and platelet dense granule abnormalities with the inner-ear abnormalities, diluted pigmentation, and lysosome secretion defects docu- mented in selected mouse mutants. We have tested several such candidate mouse mutants for platelet abnormalities. The muted and mocha pigment mutants, which have inner ear abnormalities, were found to have platelet SPD and to be animal models for CHS and HPS. Therefore, these mutants, together with some classes of HPS and CHS, represent a distinct subgroup of SPD.

MATERIALS AND METHODS

Animals. Muted pigment mice were originally obtained from The Jackson Laboratory on the CHMUlLe ch+ / +mu background. Stocks homozygous for the muted gene (mu/mu) were subse- quently bred at Roswell Park (Buffalo, NY). Mocha pigment mice were obtained from The Jackson Laboratory (Bar Harbor, ME) as the stock gr+ l +mh. Homozygous mocha (mhlmh) mice having a gray coat color and homozygous grizzled (grlgr) mice having a silverlike coat color were bred from this stock. The grizzled mice that have normal bleeding times (see Table 1) served as controls for the mocha mice in all experiments. A lighter colored stock, presumably homozygous for both mocha and grizzled genes, was also produced in these crosses, and it was found to have, like mocha, prolonged bleeding times. All other mice were obtained from The Jackson Laboratory with the exception of the silver (silsi) mutant, which was obtained from Dr Eugene Rinchik Oak Ridge National Laboratory (Oak Ridge, TN). Mice ranged in age from 1.5 to 4 months. No effect of sex on bleeding time was noted.

Bleeding time was monitored by tail bleeding as

Platelets were counted as de- scribed.” Platelets were collected for serotonin analyses and for thrombin-stimulated secretion studies by the method of Holland?’ For all other biochemical and physical studies, platelet-rich plasma was collected from citrated, undiluted blood by centrifugation at l50g for 10 minutes.

Bleeding time.

Platelet collections and counts.

2036 Blood, Vol78, No 8 (October 15). 1991: pp 2036-2044

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STORAGE POOL DEFICIENCY IN MOUSE MUTANTS 2037

Table 1. Bleeding Times in Mouse Mutants With Associated Abnormalities of the Inner Ear or of Other Physiologic Parameters

Background Strain

Bleeding Time Gene (min)

~~

C57BLl6J Normal Inner-ear abnormalities

CHMUlLe Muted (mulmu) Stockgr+l+mh Mocha (mhlmh)

C3HlHeJ Acromelanic (c’lc’) B6C3lFe Crinkled (crlcr) GLlLe Gray-lethal (g/ /g/ ) Stockgr+l+mh Grizzled (grlgr) C57BLl6J Lethal milk (lmllm) LDJlLe Mahogany (mglmg) WBB6F1 Normoblastic anemia

B6C3H Ochre (Ochl+) C57BL l10Gn Roan (Rnl+) C57BLl6J Sepia (sealsea)

Silver (silsi) C57BLl6J Taupe ( r ~ ~ ~ l t p ” ) C57BLl6J Tight skin (Tskl+) C3H101 Underwhite (uwluw) C3HlHeSn Vacuolated lens (vllvfl VlLe Waltzer (vlv) B6CBACa Weaver (wvlwv)

Other mutants

(nblnb)

2.4 f 0.4 (10)

> 15’ (13) >15* (16)

3.8 f 2.0 (4) 4.0 f 0.9 (5) 1.3 f 0.2 (3) 3.0 f 1.0 (4) 1.9 f 0.1 (4) 4.7 f 1.7 (10) 3.2 f 1.1 (3)

5.4 f 1.5 (6) 2.4 f 0.8 (6) 2.2 f 0.1 (4) 5 . 2 k 1.1 (11) 1.1 f 0.3 (6) 3.6 f 0.3 (3) 1.2 f 0.2 (3) 1.9 f 0.5 (4) 1.5 2 0.2 (6) 1.0 2 0.1 (3)

Values are expressed as the mean f SEM of the number of animals in parentheses.

*P I ,001.

Electron microscopy. Platelets, unfixed and unstained, were rapidly air-dried on carbon-coated Platelets were examined in a Hitachi H-600 transmission electron microscope (Hitachi Instruments, Santa Clara, CA) at 50 kV accelerating voltage and platelets were photographed at 12,000-fold magnification. The number of dense granules larger than 50 nm were counted in individual platelets.

Platelets were lysed in 1 mL distilled water and assayed fluorometrically for serotonin.25

Platelets were incubated with mepacrine and analyzed with a Leitz MPV-2 fluorescent microscope (E. Leitz, Inc, Rockleigh, NJ) with photomultiplier attachment as previously described.’

Platelet aggregation and adenosine triphosphate (ATP) secretion. Platelet aggregation with collagen was determined in a Chrono- Log whole blood aggregometer (model 500; Havertown, PA) as described.’6 ATP secretion was determined simultaneously by coupling with luciferin-luciferase and measuring light emitted in comparison with that produced by known concentrations of ATP.

Thrombin-stimulated platelet secretion. Platelets, preincubated with I4C-serotonin, were treated with thrombin as describedz6 to measure release of serotonin and lysosomal enzymes.

Proton pumping activity of platelet or- ganelles was measuredz6 by the initial rate of acridine orange accumulation within acidic vesicles. Estimates of acidification are expressed in arbitrary optical density units as the initial slope of ATP-dependent quenching of acridine orange.

Lysosomal enzyme concentrations in kidney and urine of testosterone-treated female mice were determined as previously described?6 Testosterone greatly ampli- fies both the synthesis and secretion of lysosomal enzymes in kidney proximal tubule cells.”

P-Glucuronidase and P-galactosidase were as-

Platelet serotonin assay.

Mepacrine uptake.

Acidifcation assay.

Urine and tissue collection.

Enzyme assays.

sayed with fluorescent methylumbelliferyl substrates?’ Protein was determined using the Bio-Rad protein assay system (Bio-Rad Labs, Richmond, CA).

I4C-serotonin was obtained from Amersham Corp (Arlington Heights, IL). Aggregation reagents were from Chrono-

Chemicals.

Log.

RESULTS

Bleeding abnormalities. Bleeding times were deter- mined in normal C57BL61J control mice, in the muted and mocha mutants with associated inner ear abnormalities, and in 17 additional mutants with abnormalities in pigmen- tation or in other physiologic parameters (Table 1). The majority of the latter mice had mutations in pigment genes.

The 2- to 3-minute bleeding time of normal mice was uniformly increased to greater than 15 minutes in all homozygous muted and mocha mice. This increase was true for both sexes.

All the other mutants tested had normal bleeding times. In the case of the mahogany and silver mutants, there were two mice in each group that had prolonged bleeding times (> 15 minutes) in the original tests. However, in two repeat tests on the same mice, the bleeding times had returned to normal.

Several physiologic tests were per- formed to characterize the prolonged bleeding times in the muted and mocha mutants. A lowered platelet count is not responsible for the bleeding tendencies. Similar counts of 1.2 x lo9 and 1.3 x lo9 plateletslml (mean of four mice) were found in the mu/+ and mulmu mice, respectively. Likewise, similar counts of 1.17 x lo9 and 1.10 x lo9 plateletslml were obtained in the mocha control and mhlmh mice, respectively.

Because platelet storage pool deficiency has been found to be the cause of the prolonged bleeding times in other mouse pigment mutation^'^^*'^ including the pallid mutant’ with its associated inner-ear abnormalities, muted and mocha platelets were examined for characteristics of stor- age pool deficiency.

A principal chemical constituent of platelet dense gran- ules, serotonin, was found to be highly deficient in both muted and mocha platelets (Table 2) . In mulmu platelets, serotonin levels were less than 8% those of control (mu/ +), while in mhlmh platelets these values were less than 6% of normal (control-mocha) levels. Another major component stored in platelet dense granules is ATP. When collagen was added to normal platelets, ATP was secreted in large amounts (Fig 1). However, minimal or no releasable ATP was detected when either low (1 pg/mL) or high (4 pg/mL) concentrations of collagen were added to mulmu or mhlmh platelets, consistent with a deficiency in the granule storage pool of adenine nucleotides.

A definitive morphologic indication of platelet storage pool deficiency is a decreased number of identifiable granules in platelet whole mounts as determined by elec- tron micros~opy.~~ The lack of visible dense granules with a clear outline in typical mulmu and mhlmh platelets is illustrated in Fig 2. The number of dense granules per platelet in normal mice covers a wide range: from 0 to 20

Physiologic analyses.

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2038 SWANK ET AL

Table 2. Characteristics of Dense Granules of Normal and Mutant Platelets as Determined by Fluorescence Microscopy of Mepacrine-Labeled Platelets and by Serotonin Analyses

Relative Serotonin GranuleslPlatelet Flashes/Platelet FlashesIGranule Intensity (mv) (pgI1O’ platelets)

mu/+ 5.84 f 0.32 (25) 4.44 f 0.48 (25) 0.76 39.0 f 2.7 (33) 2.09 f 0.052 (3) mulmu 5.53 f 0.46 (26) 0.84 2 0.21 (26)’ 0.15’ 25.8 f 2.3 (20)t 1.68 f 0.017 (8)’ Control mocha 5.52 f 0.31 (23) 5.00 f 0.33 (23) 0.90 23.1 f 2.8 (25) 1.50 f 0.10 (3) mhlmh 5.16 f 0.34 (24) 0.75 f 0.19 (26)’ 0.14’ 13.2 f 1.5 (28)$ 0.08 f 0.02 (3)’

Values represent the mean f SEM of the number of determinations indicated in parentheses. Relative intensity per platelet was determined before commencement of the flashing phenomenon. The values forflasheslgranule were determined by dividing the flashes per platelet by the granules per platelet.

‘f I ,001. t P 5 .01. SP I .05.

granules per platelet, with a mean of 8 (Fig 3). In contrast, the great majority of both mulmu and mhlmh platelets had no visible granules. A few mutant platelets had up to three granules per platelet. No apparent differences were noted in the size of mutant and normal platelets. The small ( < 30 nm) beadlike particles visible in the muted platelet (Fig 2A) were observed in other platelets of both normal and mocha platelets. They have previously been observed6 in both normal and mutant mice. Their significance is unknown.

Another test of the quality of the intragranular contents and environment of platelet dense granules is to measure their ability to incorporate the fluorescent dye mepacrine. On incubation with platelets, this basic dye is preferentially incorporated into platelet dense granules and on illumina- tion with ultraviolet light a flashing phenomenon, thought to be due to lowered quenching of fluorescence when mepacrine is released from platelet dense granules into the cytosol, occurs.2s In SPD patients and in animal models with SPD, this flashing phenomenon is typically absent or much reduced, presumably due to an abnormality in the environ- ment within dense granules.

A normal number of mepacrine-labeled dense granules were observed in muted and mocha platelets (Table 2). However, the flashing phenomenon was reduced more than fivefold in each mutant. Also, the amount of mepacrine incorporated into mutant dense granules, as measured by millivolt relative intensity, was also significantly decreased. Both results suggest an abnormal intragranular environ- ment.

Storage pool deficient platelets often have abnormal collagen-mediated aggregation, particularly at low collagen concentrations. In fact, a decreased extent of aggregation was noted for each mutant at both high and low collagen concentrations on impedance analysis in whole blood aggre- gometry (Fig 1). The reduction in aggregation, relative to normal platelets, was particularly pronounced at low (1 p,g/mL) collagen concentrations. Collagen-induced aggre- gation and release of ATP is also reduced in human HPS.’6*29

Platelet SPD in murine mutants and in humans with SPD and CHS is characterized by abnormalities in form or function of other intracellular organelles, including melano- somes and lysosomes. Both muted and mocha have pigmen- tation abnormalities as manifested by their diluted coat

color. In mouse SPD mutants the lysosome defect is conveniently monitored by quantitating the secretion of kidney lysosomal enzymes into urine.” In the case of the muted mutant, kidney concentrations of all three lysosomal enzymes ( p-glucuronidase, p-galactosidase, and p-glucosi- dase) were significantly increased relative to control mu1 + mice (Table 3). The cause of these elevated kidney lyso- somal enzyme levels is a decreased rate of enzyme secretion from kidney into urine (Table 4). Consistent with this interpretation is the fact that lysosomal enzyme levels were normal in liver and platelets in which constitutive lysosomal enzyme secretion is absent or minimal. Similarly, lysosomal enzyme levels were elevated in kidneys of mocha mice (Table 3), and this elevation was associated with a de- pressed rate of secretion into urine (Table 4). Control and mutant mice are known to respond similarly to testosterone because the degree of kidney hypertrophy, an independent measure of testosterone sensitivity,” is equal in muted or mocha mutant mice and their appropriate control mice (R.T.S., data not shown). The urinary secretion of glucuroni- dase in mu/+ is higher than in control mocha (Table 4) because mu/+ mice contain a gene GuP that causes a twofold to threefold increased rate of synthesis of kidney glucuronidase in response to testosterone.”’

Several platelet subcellular organelles including lyso- somes are secreted when platelets are treated with various agonists including thrombin. Lysosomal enzyme secretion from platelets of SPD patients is often depressed, especially in platelets from HPS patients.” Secretion of glucuronidase from mulmu platelets was nominally decreased at both .25 and 2.5 U thrombin, although the decrease was statistically significant only at 2.5 U thrombin (Table 5). Galactosidase secretion was 60% to 70% of normal at both thrombin concentrations. The depressed secretion was not changed by inclusion of 10 KmollL adenosine diphosphate (ADP), which is known to correct the abnormal secretion in human HPS platelets.29332 An abnormal thrombin-induced secretion of lysosomal enzymes was even more apparent with mocha platelets (Table 5). Secretion of both lysosomal enzymes was only 30% to 45% of normal, and this depressed secretion was not corrected by addition of ADP.

Secretion of a dense granule marker, serotonin, was also significantly decreased with both mutant platelets (Table 5). The most striking feature of serotonin secretion from

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STORAGE POOL DEFICIENCY IN MOUSE MUTANTS 2039

t h

u) E 5.0- Y 5 5 4.0- .- c g,

8 Q)

3.0-

2.0 -

1.0-

A

1 2 3 4 5 6 7 e Time (m id

B

t v s h m E

E 0 0 0

.- c

8

I I I I I I 4 2 3 4 5 6 7

Time (mid

D

t - m E v s C 0

0 0

.- c

s a

3

n I- 1.2 a cn 9)

0 3.4 E

C Q)

- Y

3.6 f 3

3 1.0 I

1

a3 C 3.8 -E

4. 2

Time (min) Time (mid Fig 1. Platelet aggregation and release of ATP examined in mutant mulmu, mhlmh, and normal platelets in response to collagen. Platelet

aggregation was measured by impedance in whole blood in response to 1 pg/mL (A and C) and 4 pglmL (B and D). ATP release was simultaneously determined by luminescence methods. The small arrow indicates the time of addition of collagen.

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2040

56 52 48

44-

40- 36- 32

a 28- 0 24- 0)

‘0 20- b 46-

0 42-

5 8 - n

L - ).

SWANK ET AL

- - -

-

C

Fig 2. Electron micrographs of whole-mounted platelets from mu/ mu (A), normal (B), and mhlmh (C) mice. A typical dense granula of a normal platelet is indicated by the arrow. The bar represents 1 Km.

mutant platelets is that large quantities (approximately 40%) were released in the absence of thrombin stimulation during the 3-minute incubation, a result that emphasizes the defective storage mechanism in these granules.

A possible explanation for the abnormal secretion of lysosomal enzymes from mutant lysosomes is a flaw in lysosomal acidification. Hohman and Bowers” provided evidence that a pH-dependent binding of lysosomal en- zymes to organellar membranes influences their secretion rate. Also, the three organelles (melanosomes, lysosomes, and platelet dense granules) that are defective in the mouse pigment mutants have acidic interiors in common.”’M Ac- cordingly, the proton pumping ability of acidic organelles of normal and mutant extracts was compared by measuring (Table 6) the initial rate of accumulation of acridine orange within the vesicles?‘ No difference was apparent in the ATP-stimulated acidification activity of extracts of normal and mutant platelets.

A characteristic of human HPS is that ceroidlike pigment is present in several cell types including macrophages and kidney proximal tubule cells.’3 Higher-than-normal levels of autofluorescence, typical of ceroidlike pigment, were, in fact, detected in proximal tubules when frozen sections of kidneys of muted and mocha mice were examined by ultraviolet fluorescence microscopy. Also, retinal pigment epithelial cells were examined for the presence of giant granules that are characteristic of CHS. Granules of these cells were slightly larger than normal in the muted and mocha mutants, but they were much smaller than the giant granules observed in retinal pigment epithelial cells of the

beige mouse, which is an animal model for CHS. This finding suggests that muted and mocha mutants are more appropriate models for the HPS than for the CHS form of SPD.

DISCUSSION

These experiments show that platelet SPD is associated with inherited inner-ear abnormalities and pigment dilu-

24

20

16 cn

Q)

a - 4 0

t

32 12

= 0

& o

c

0 E 12

= 0 3

4 c

Muted Control

A - 0 2 4 6 0 10 12 1416 Dense Granules per Platelet

Dense Granules Fer Plat&

Flg 3. Histograms of numbers of dense granules in platelets from control and mutant mice. The numkr of platelets analyzed wau 30 in the cases of mu/mu and muted control mice and 60 in the cases of mh/ mh and mocha control mice.

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STORAGE POOL DEFICIENCY IN MOUSE MUTANTS 2041

Table 3. Concentrations of Lysosomal Enzymes in Kidney, Liver, and Platelets of Testosterone-Treatad Control and Mutant Mice

p-Glucuronidase p-Galactosidase p-Glucosidase

Kidney (Ulg) mu/+ 160 f 10 mulmu 207 ? 16, Mocha control 74.9 f 3.2 mhlmh 156 f 9.4*

Liver (Ulg) mu/+ 24.6 f 0.38 mulmu 30.0 f 4.4 Mocha control 16.6 f 0.61 mhlmh 16.0 f 0.50

Platelets (U/lOs platelets) mol+ 0.0349 f 0.0037 mulmu 0.0429 f 0.0044 Mocha control 0.0642 f 0.0084 mhlmh 0.0753 f 0.0077

24.8 f 1.7 55.0 2 1.8* 33.5 f 0.73 - 58.9 f 0.73 -

0.48 f 0.03 0.64 f 0.017

17.1 f 1.7 13.9 f 1.2 10.6 f 0.55 - 14.4 f 0.55 -

1.73 f 0.06 1.78 f 0.12

0.0507 f 0.0054 - 0.0599 f 0.0076 - 0.0881 f 0.0079 - 0.0698 f 0.0065 -

Each value is the mean f SEM of four mice (@-glucuronidase and

*P 5 ,001. tP s .02.

@-galactosidase) or three mice (@-glucosidase).

tion in the mouse pigment mutants muted and mocha. Previous studies7 had documented a similar association in the pallid pigment mutant. No inner-ear abnormalities are apparent in nine other independent mouse mutants with SPD.7*m326237 Therefore, the muted, mocha, and pallid mu- tants represent a distinct subgroup of SPD.

Comparable associations have been described in humans with HPS" and CHS.I7 Selected HPS patients have platelet SPD, inner-ear abnormalities, and diluted pigmentation. The similarities of HPS patients and the previous three mouse mutants extend to other feature^,'^^'^ including decreased secretion of lysosomes from platelets and ceroid pigment deposition. Inner-ear abnormalities and reduced inner-ear pigmentation have also been described in the human Waardenburg syndrome." However, in this case the defect may be more closely related to the white spotting pigment condition, in which the formation of melanocytes in the neural crest or their migration to target organs is disturbed, rather than to the dilution pigment syndromes. Auditory anomalies are also found in human albinos." To

Table 4. Daily Secretion of Lysosomal Enzymes Into Urine of Control and Mutant Mice

p-Glucuronidase @-Galactosidase (Ulmouse) (Utmouse)

mu1 + 11.6 f 0.85 1.97 f 0.13 mulmu 6.43 +' 0.79' 1.10 f 0.10t Control mocha 5.5 2 0.60 2.69 f 0.34 mhlmh 1.08 f 0.46* 0.80 2 0.1 1 *

Each metabolism cage contained four mice that had been treated with testosterone far 22 days. Values represent the mean f SEM of the mean of five determinations on consecutive days. *P 5 .01. tP 5 .02.

our knowledge, it is unknown whether structural abnormal- ities of the otoliths and other organs of the inner ear such as those found in pallid, muted, and mocha are present in human CHS and HPS. It is known that the auditory defects in CHS and HPS are often accompanied by visual anoma- lies.'*

In inner-ear structural abnormalities are also found in several white spotting hypopigmented genotypes. Similarly, an association of inner-ear structural abnormali- ties with hypopigmentation has been found in cats, dogs, and

The mechanism of association of pigment and inner-ear abnormalities is probably related to the presence of mela- nin in the normal inner ear." The amount of pigment in the inner ear is correlated with the amount in the iris and is absent or reduced in albinos. It has been postulated" that the paucity of pigment in the inner ear of CHS patients may be related to a neuronal cascade of changes that affect the auditory pathways. The pallid and mocha mutants have abnormalities of inner ear pigmentationa in addition to the structural defects in otoliths and other organs of the inner ear. No studies on inner ear pigmentation in muted mice have been reported. Melanin of the inner ear may be a reservoir4' for the binding of metals such as manganese, which is present in increased concentrations in pigmented tissues4' where it may function in the biosynthesis of sulfated mucopolysaccharides.42 This metal binding would, of course, be reduced in mutants with reduced or absent pigment.

It is interesting that the congenital otolith defects, but not the abnormal pigmentation, are prevented in the cases of offspring of the pallid and mocha mutants by supplemen- tation of the diet of pregnant females with metals such as manganese.a243 One of us has found (E.K.N., data not shown) that dietary manganese supplementation does not correct the abnormal kidney lysosomal enzyme secretion in offspring of the pallid mutant. Similarly, no abnormalities in 54Mn binding proteins were apparent when Western blots" of platelet extracts of the muted mocha and pallid mutants were examined.

Previous s t ~ d i e s ~ ~ ~ ' , ~ ~ have documented that genes which cause pigment dilution often have associated platelet SPD and lysosome defects. The combined results of all stud-

including this report, are that 12 of 34 mouse pigment dilution mutations have an associated prolonged bleeding time caused by SPD. These 12 genes are usually qualitatively and/or quantitatively distinct in their effectsx and are found at distinct chromosomal sites. Therefore, it is apparent that many genes control the biogenesis and/or processing of at least three subcellular organelles including melanosomes, lysosomes, and platelet dense granules and that a single gene can simultaneously affect all three subcellular organelles. In both mice and accord- ingly, it is likely that a large number of genes can cause SPD. However, a final answer to the question of whether the phenotypically disparate'2.'6 classes of human HPS are caused by different primary genes or by a single HPS gene, modified by the inherently heterogeneous genetic back-

ies,7.20,26,37

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2042 SWANK ET AL

Table 5. Thrombin-Stimulated Secretion of Lysosomal Enzymes and Serotonin From Platelets of Control and Mutant Mica

Thrombin (U)

0 .25 2.5 2.5 + ADP

Secretion (K total)

p-Glucuronidase mu/+ 2.7 2 0.60 19 f 3.2 32 f 3.5 32 f 2.9

22 f 1.2t mulmu 1.8 f 0.62 1 1 f 2.7 Mocha control 3.7 f 0.9 39 f 3.9 45 f 3.6 48 f 4.0 mhlmh 4.3 f 2.4 10 f 1.1* 16 f 3.5t 18 f 1.8*

38 f 2.7 mu/+ 5.2 f 0.77 29 f 3.2 mulmu 5.0 f 1.8 17 f 1.5t 26 f 1.6* 28 f 1.6t Mocha control 5.3 2 2.4 45 f 3.2 53 f 2.6 54 f 4.5 mhlmh 5.9 f 1.5 1 1 f 1.2* 25 f 3.1' 27 f 3.0t

mu/+ 5.2 f 0.20 82 f 1.0 87 2 0.4 88 f 0.2 mulmu 37 f 5.0$ 46 f 3.0$ 50 f 2.8$ 57 f 1.3$ Mocha control 2.6 f 0.8 86 f 1.0 88 f 0.6 88 f 1.3 mhlmh 44 2 6.1* 54 f 0.9* 56 f 2.l* 60 f 0.9"

17 f 1.3t

p-Galactosidase 36 f 1.6

'Cserotonin

Platelets (1 x 10'1mL) were treated with the indicated units of thrombin for 3 minutes at 37°C or with thrombin + 10 pmol1LADP. The reaction was stopped with 2.5 nmol1mL thromstop (American Diagnostic Inc, Greenwich, CT). Values represent the mean f SEM of determinations on four separate pools of platelets with each pool derived from four mice. Total cpm "C-serotonin initially incorporated in mu/+, mulmu, mocha control, and mhlmh platelets were 2,800,917,2,730, and 632, respectively, per 10' platelets.

*P 5 .01. tP 5 .05. SP 5 .001.

ground among different humans, will likely require molecu- lar analysis of the human HPS gene(s).

The dense granule deficiency is quite severe in both muted and mocha platelets. The deficiency in mocha platelets is, in fact, even more severe than that of the sandy mutant,26 which previously had the lowest recorded number of dense granules of any mouse mutant.

It is interesting that the tight skin gene causes no prolongation (Table 1) of bleeding time despite the fact that it has abnormalities" in collagen, which is a major protein of blood vessels. Likewise, the vacuolated lens mutation, which causes the accumulation of vacuoles in the developing lens,& apparently has no major effect on subcel- lular organelles of platelets. No genes that cause pigment intensification in mice (such as sombre, mahoganoid?' and mahogany [Table 11) have been found to cause hematologic abnormalities. Also, not all mouse pigment genes associ- ated with pigment dilution cause prolonged bleeding times. Nine in this study (acromelanic, gray lethal, grizzled, ochre, roan, silver, taupe, underwhite, and sepia) have normal

Table 6. ATP-Stimulated Acidification Activity of Extracts of Normal and Mutant Platelets

bleeding times. Furthermore, not all mouse genes associ- ated with abnormal metal metabolism have hemostatic defects because the crinkled4' and lethal milk48 mutations, which are associated with the abnormal metabolism of copper and zinc, respectively, have normal bleeding times (Table 1). Finally, inherited inner-ear abnormalities in the mouse are not invariably associated with prolonged bleed- ing times, as the waltzer (vol/vol) mutation of this study had a normal bleeding time (Table 1) despite known degenera- tion of the organ of Corti, spiral ganglion, stria vascularis, and saccular ma~ula.4~

The molecular mechanisms by which platelet SPD and inner-ear abnormalities are associated in the pallid, muted, and mocha mutants is uncertain. Studies in yeast have shown that a large number of genes are involved in organelle biogenesis. For example, at least 23 genes" are involved in the secretion of organelles from yeast and a minimum of 49 distinct genes whose products are required to sort, transport, and/or retain lysosomal proteins have been identified?l The molecular structures and/or func- tions of several of these yeast genes have recently been

Initial Slopelmg Protein Genotype (xi031

Muted control 6.1 2 0.53 (4) mulmu 8.1 f 0.80 (6) Mocha control 6.3 2 0.3 (6) mhlmh 5.9 2 0.9 (3)

The initial slope is expressed as the absorbance 492 nm-540 nm per minute. Numbers in parentheses are the number of separate determina- tions made on platelet extracts pooled from three mice.

ACKNOWLEDGMENT

Cheryl Mrowczynski supplied secretarial service and AI Cairo provided electron microscopic analysis of platelets. We thank Jie Wang for technical assistance and Dr Dennis Stephenson for assistance in microscopic analysis of pigment granules in retinal pigment epithelial cells. Hope Sweet, Dr Muriel Davisson, and Dr Jane Barker of The Jackson Laboratories and Dr Gene Rinchik of Oak Ridge National Laboratory generously supplied the various mutant mice for this study.

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STORAGE POOL DEFICIENCY IN MOUSE MUTANTS

REFERENCES

2043

1. Lyon MF, Meredith R: Muted, a new mutant affecting coat colour and otoliths of the mouse, and its position in linkage group XIV. Genet Res 100:163,1969

2. Lane PW, Deol MS: Mocha, a new coat color and behavior mutation on chromosome 10 of the mouse. J Hered 65362,1974

3. Lyon MF, Searle AG (eds): Genetic Variants and Strains of the Laboratory Mouse (ed 2). New York, NY, Oxford, 1989, p 274

4. Lyon M F Absence of otoliths in the mouse: An effect of the pallid mutant. J Genet 51:638,1953

5. Lyon M F The developmental origin of hereditary absence of otoliths in mice. J Embryo1 Exp Morpho1 3:230,1953

6. Novak EK, Swank R T Lysosomal dysfunctions associated with mutations at mouse pigment genes. Genetics 92:189,1979

7. Novak EK, Hui S-W, Swank RT: Platelet storage pool deficiency in mouse pigment mutations associated with seven distinct genetic loci. Blood 63536,1984

8. Novak EK, McGany MP, Swank R T Correction of symptoms of platelet storage pool deficiency in animal models for Chediak- Higashi syndrome and Hermansky-Pudlak syndrome. Blood 6 6 1196,1985

9. Reddington M, Novak EK, Hurley E, Medda C, McGarry MP, Swank R T Immature dense granules in platelets from mice with platelet storage pool disease. Blood 69:1300,1987

10. McGarry MP, Novak EK, Reddington M, Swank R T Effects of mixed chimeric bone marrow repopulation on platelet storage pool-associated bleeding defects in mouse mutants. Exp Hematol 18:1174,1990

11. Chediak-Higashi syndrome, in Klebanoff SJ, Clark RA (eds): The Neutrophil Function and Clinical Disorders. New York, NY, North Holland, 1978, p 735

12. Witkop CJ, Quevedo WC, Fitzpatrick TB, King R A Albi- nism, in Scriver CR, Beaudet AL, Sly WS, Valle D (eds): The Metabolic Basis of Inherited Disease (ed 6). New York, NY, McGraw-Hill, 1989, p 2905

13. Depinho RA, Kaplan KL: The Hermansky-Pudlak syn- drome, report of three cases and review of pathophysiology and management considerations. Medicine 64192,1985

14. Nieuwenhuis HK, Akkerman J-WN, Sixma JJ: Patients with a prolonged bleeding time and normal aggregation tests may have storage pool deficiency: Studies on one hundred six patients. Blood 70620,1987

15. Israels SJ, McNicol A, Robertson C, Gerrard JM: Platelet storage pool deficiency: Diagnosis in patients with prolonged bleeding times and normal platelet aggregation. Br J Haematol 75:118, 1990

16. Weiss HJ, Witte LD, Kaplan KL, Lages BA, Chernoff AN, Goodman DS, Baumgartner H R Heterogeneity in storage pool deficiency: Studies on granule-bound substances in 18 patients including variants deficient in a-granules, platelet factor 4, p-throm- boglobulin and platelet-derived growth factor. Blood 541296,1979

17. Creel D, Boxer LA, Fauci AS: Visual and auditory anoma- lies in Chediak-Higashi syndrome. Electroencephalogr Clin Neuro- physiol55:252, 1983

18. Garber SR, Turner CW, Creel D, Witkop CJ Jr: Auditory system abnormalities in human albinos. Ear Hear 3:207,1982

19. Lorincz AL: Disturbances of melanin pigmentation, in Moschella SL, Hurley HJ (eds): Dermatology (vol 2, ed 2). Philadelphia, PA, Saunders, 1985, p 1290

20. Novak EK, Sweet HO, Prochazka M, Parentis M, Soble R, Reddington M, Cairo A, Swank RT: Cocoa: A new mouse model for platelet storage pool deficiency. Br J Haematol69:371,1988

21. Dejana E, Callioni A, Quintana A, Gaetano G: Bleeding time in laboratory animals. 11. A comparison of different assay conditions in rats. Thromb Res 15191,1979

22. Brecher G, Cronkite EP: Morphology and enumeration of human blood platelets. J Appl Physiol3:365,1950

23. Holland JM: Serotonin deficiency and prolonged bleeding in beige mice. Proc SOC Exp Biol Med 151:32,1976

24. Hui S-W, Costa J L Topography and thickness of air-dried human platelets measured by correlative transmission and scan- ning electron microscopy. J Microsc 115:203,1979

25. Crosti P, Lucchelli PE: An easy method to determine the serotonin content of human platelets. J Clin Pathol15:191,1962

26. Swank RT, Sweet HO, Davisson MT, Reddington M, Novak E K Sandy: A new mouse model for platelet storage pool defi- ciency. Genet Res (in press)

27. Brandt EJ, Elliott RW, Swank R T Defective lysosomal enzyme secretion in kidneys of Chediak-Higashi (beige) mice. J Cell Biol67:744,1975

28. Lorez HP, Da Prada M, Pletscher A Flashing phenomenon in blood platelets stained with fluorescent basic drugs. Experientia 31593, 1975

29. Rendu F, Maclouf J, Launay J-M, Bornot C, Levy-Toledano S, Tanzer J, Caen J: Hermansky-Pudlak platelets: Further studies on release-reaction and protein phosphorylations. Am J Hematol 25165,1987

30. Swank RT, Paigen K, Davey R, Chapman V, Labarca C, Watson G, Ganschow R, Brandt EJ, Novak E: Genetic regulation of mammalian glucuronidase, in Greep RO (ed): Recent Progress in Hormone Research (~0134). San Diego, CA, Academic, 1978, p 401

31. Lages B: Studies on storage mechanisms in the dense granules: Storage pool deficient platelets, in Holmsen H (ed): Platelet Responses and Metabolism, (vol 11). Boca Raton, FL, CRC, 1987, p 135

32. Lages B, Dangelmaier CA, Holmsen H, Weiss HJ: Specific correction of impaired acid hydrolase secretion in storage pool deficient platelets by adenosine diphosphate. J Clin Invest 81: 1865, 1988

33. Hohman TC, Bowers B: Hydrolase secretion is a conse- quence of membrane recycling. J Cell Biol98:246,1984

34. Anderson RG, Orci L Aview of acidic intracellular compart- ments. J Cell Biol106:539,1988

35. Wolff K, Schreiner E: Epidermal lysosomes. Arch Dermatol 101:276,1970

36. Dean GE, Fishkes H, Nelson PJ, Rudnick G: The hydrogen ion-pumping adenosine triphosphatase of platelet dense granule membrane. J Biol Chem 2599569,1984

37. Ahmed F, Lundin L-G, Shire JGM: Lysosomal mutations increase susceptibility to anesthetics. Experientia 45:1133,1989

38. Deol MS: The relationship between abnormalities of pigmen- tation and of the inner ear. Proc R SOC Lond A 175:201,1987

39. Schrott A, Spoendlin H: Pigment anomaly-associated inner ear deafness. Acta Otolaryngol (Stockh) 103:451,1987

40. Erway LC: Genetic and developmental implications for trace metal metabolism from mutant and inbred strains of animals, in Rennert OM, Chan W-Y (eds): Metabolism of Trace Metals in Man (vol I). Boca Raton, FL, CRC, 1984, p 17

41. Cotzias GC, Papavasiliou PS, Miller ST: Manganese in melanin. Nature 201:1228,1964

42. Shrader RK, Erway LC, Hurley Ls: Mucopolysaccharide synthesis in the developing inner ear of manganese-deficient and pallid mutant mice. Teratology 8:257,1973

For personal use only.on January 4, 2019. by guest www.bloodjournal.orgFrom

2044 SWANK ET AL

43. Envay LC, Fraser AS, Hurley LS: Prevention of congenital otolith defect in pallid mutant mice by manganese supplementa- tion. Genetics 67:97,1971

44. Maruyama K, Mikawa T, Ebashi S: Detection of calcium binding proteins by "Ca autoradiography on nitrocellulose mem- branes after sodium dodecyl sulfate gel electrophoresis. J Biochem 95511,1984

45. Jiminez SA, Williams CJ, Myers JC, Bashey RI: Increased collagen biosynthesis and increased expression of type I and type I11 procollagen genes in tight-skin (Tsk) mouse fibroblasts. J Biol Chem 261:657,1986

46. Dickie MM: Mouse News Lett 36:39,1967 47. Keen CL, Hurley LS: Superoxide dismutase activity in the

crinkled mutant mouse: Ameliorative effects of dietary copper supplementation. Proc SOC Exp Biol Med 162152,1979

48. Piletz JE, Ganschow RE: Zinc deficiency in murine milk underlies expression of the lethal milk (lm) mutation. Science 199:181,1978

49. Deol MS: The anatomy and development of the mutants pirouette, shaker-1 and waltzer in the mouse. Proc R SOC Lond [Biol] 145206,1956

50. Novick P, Field C, Schekman R: Identification of 23 comple- mentation groups required for post-translational events in the yeast secretory pathway. Cell 21:205, 1980

51. Rothman JH, Yamashiro CT, Kane PM, Stevens TH: Protein targetting to the yeast vacuole. Trends Biochem Sci 14:347, 1989

52. Bohni PC, Deshaies RJ, Schekman RW: SECII is required for signal peptide processing and yeast cell growth. J Cell Biol 106:1035,1988

53. Kepes F, Schekman R: The yeast SEC53 gene encodes phosphomannomutase. J Biol Chem 263:9155,1988

54. Herman PK, Stack JH, De Modena JA, Emr SD: A novel protein kinase homolog essential for protein sorting to the yeast lysosome-like vacuole. Cell 64:425,1991

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1991 78: 2036-2044  

RT Swank, M Reddington, O Howlett and EK Novak and mochaabnormalities of the inner ear in the mouse pigment mutants muted Platelet storage pool deficiency associated with inherited 

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