Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4856-4863
Hematopoietic Cells From 
-Spectrin-Deficient Mice Are Sufficient
to Induce Thrombotic Events in Hematopoietically Ablated Recipients
By
Nancy J. Wandersee,
John C. Lee,
Tamma M. Kaysser,
Roderick T. Bronson, and
Jane E. Barker
From The Jackson Laboratory, Bar Harbor, ME; the School of Medicine,
University of Connecticut, Farmington, CT; Motorola Corp, Schaumberg,
IL; and the Tufts University School of Veterinary Medicine, Boston, MA.
 |
ABSTRACT |
Thrombotic events are life-threatening complications of human
hemolytic anemias such as paroxysmal nocturnal hemoglobinuria, sickle
cell disease, and thalassemia. It is not clear whether these events are
solely influenced by aberrant hematopoietic cells or also involve
aberrant nonhematopoietic cells. Spherocytosis mutant
(Spna1sph/Spna1sph; for simplicity
referred to as sph/sph) mice develop a severe hemolytic anemia
postnatally due to deficiencies in 
-spectrin in erythroid and other
as yet incompletely defined nonerythroid tissues. Thrombotic lesions
occur in all adult sph/sph mice, thus providing a
hematopoietically stressed model in which to assess putative causes of
thrombus formation. To determine whether hematopoietic cells from
sph/sph mice are sufficient to initiate thrombi, bone marrow
from sph/sph or +/+ mice was transplanted into mice with no
hemolytic anemia. One set of recipients was lethally irradiated; the
other set was genetically stem cell deficient. All mice implanted with
sph/sph marrow, but not +/+ marrow, developed severe anemia and histopathology typical of sph/sph mice. Histological
analyses of marrow recipients showed that thrombi were present in the
recipients of sph/sph marrow, but not +/+ marrow. The
results indicate that the -spectrin-deficient hematopoietic cells
of sph/sph mice are the primary causative agents of the
thrombotic events.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
THROMBOSIS IS clinically relevant in
humans with hematopoietic diseases, most notably paroxysmal nocturnal
hemoglobinuria (PNH), sickle cell disease (SCD), and
thalassemia.1-5 In patients with hereditary spherocytosis
(HS), there is one report of thrombosis.6 However, because
expression of cytoskeletal genes is not restricted to erythroid cells,
it is not clear whether the thrombotic events are primarily due to
aberrant cells of the hematopoietic lineage or if there are also
contributions from nonhematopoietic tissues.
Deficiencies of - and 
-spectrin, ankyrin, and band 3, essential
structural components of the red blood cell (RBC)
cytoskeleton,7 are responsible for most cases of HS in
humans.8,9 In the absence of any of these cytoskeletal
components, a severe hemolytic anemia can arise due to increased
osmotic fragility of the erythrocytes.10,11 The severity of
disease in humans is quite variable, ranging from relatively mild
to very severe clinical manifestations.8,9 Recently,
hydrops fetalis has also been associated with HS due to - or
-spectrin defects.12,13
Known HS syndromes in laboratory mice (Mus) can be attributed
to autosomal recessive mutations at three separate loci. Mice with
normoblastosis (Ank1nb/Ank1nb; for
simplicity, referred to as nb/nb) are ankyrin deficient, jaundiced (Spnb1ja/Spnb1ja; for
simplicity referred to as ja/ja) mice are -spectrin
deficient, and spherocytic
(Spna1sph/Spna1sph; for simplicity
referred to as sph/sph) mice are -spectrin
deficient.14-16 HS mice lacking RBC band 3 have been
generated by disruption of the gene through homologous
recombination.17,18 All mice with defective cytoskeletons
develop a severe hemolytic anemia within hours of birth.19
The spherocytic erythrocytes are extremely short-lived (1 day v
48 days in normal mice), and reticulocytes comprise 75% to 95% of the
peripheral hemoglobinized cells.14 Pathophysiologic effects
of the hemolytic anemia include cardiomegaly, hepatomegaly,
splenomegaly, deposition of iron in the liver and kidney, and premature
death.20
HS disease manifestations in nonerythroid tissues may not be entirely
due to secondary effects of the hemolytic anemia. Expression of
erythroid cytoskeletal genes is not limited to RBCs. It is, in fact,
not yet clear how widely distributed transcripts of any of these
cytoskeletal components may be. Isoforms of both - and -spectrin
have been reported in brain21-24 and eye.24 In
addition, -spectrin has been detected in skeletal and heart
muscle,22,23 as well as in kidney.25 Ankyrin
and band 3 have tissue distributions similar to
-spectrin.26-28 Deficiency of ankyrin in the cerebellum of nb/nb mice, and not the hemolytic anemia, causes latent
Purkinje cell degeneration concomitant with the development of a
neurologic phenotype.28
Recently, we detected thrombotic lesions in adult mice with
-spectrin, -spectrin, and ankyrin deficiencies but not in any of
their normal littermates.20 All adult sph/sph
mice had thrombotic and embolic lesions in the heart and brain. By
contrast, only 15% of ja/ja mice had heart lesions and 38%
had brain lesions.20 Twenty percent of nb/nb mice
had both heart and brain lesions (J.E. Barker, unpublished
data). A high incidence of thrombosis has also been
detected in mice homozygous for one of the band 3 knockouts.29 Mice with HS provide a model for thrombosis in the context of an underlying hemolytic anemia. Because erythroid cytoskeletal genes are expressed in nonhematopoietic tissues, we wished
to determine whether thrombogenesis in sph/sph mice involved
only aberrant hematopoietic cells. In the present report, we show that
sph/sph hematopoietic cells are sufficient to induce thrombus
formation when transferred to hematopoietically ablated mice that have
normal cytoskeletal gene expression in nonhematopoietically derived
cell types.
| |
MATERIALS AND METHODS |
Mice used for bone marrow transplantation (BMT).
Normal and mutant mice were maintained on both the WB/Re (WB) and
C57BL/6J (B6) backgrounds. F1 hybrid (WBB6F1) mutant mice were
generated by mating WB-sph/+ × B6-sph/+ mice.
Normal WBB6F1 mice were generated by mating WB-+/+ × B6-+/+ mice.
Donors for BMT experiments were WBB6F1-sph/sph (henceforth
referred to as sph/sph) and WBB6F1-+/+ (henceforth referred
to as +/+) females. These mice were homozygous for the B isoform of the
ubiquitously expressed enzyme glucose phosphate isomerase 1 (GPI1;
Gpi1b/Gpi1b) and heterozygous for
-globin haplotype (Hbbd/Hbbs).
Recipients were irradiated male and female WB(B6.CAST-
Gpi1a)F1-+/+ (henceforth referred to as
+/+a) and genetically stem cell-deficient
WBB6F1-W/Wv (henceforth referred to as
W/Wv) mice. The +/+a recipients were
obtained by mating wild-type
WB-Gpi1b/Gpi1b,
Hbbd/Hbbd to congenic
B6.CAST-Gpi1a/Gpi1a,
Hbbs/Hbbs mice and were therefore
heterozygous for both GPI1
(Gpi1a/Gpi1b) and -globin haplotype
(Hbbd/Hbbs). Donor and +/+a
host cells were easily differentiated through analyses of GPI1AB versus
BB ratios in peripheral blood cells. One hundred percent GPI1BB was
indicative of complete conversion to donor cells. The W/Wv recipient mice had the same genetic markers
at the Gpi1 and Hbb loci as the donors. Donor
repopulation of the W/Wv hosts was assessed
by the cure of the host macrocytic, normochromic anemia after
implantation of +/+ cells and by the worsening of the anemia and
assumption of typical sph /sph histopathology after implantation of sph/sph cells. Mice were housed and cared for according to AAALAC specifications.
To ensure recipient survival during the initial amplification of donor
cells, two modifications were made to our existing protocols.30 First, the irradiation-myeloablated
+/+a recipients were injected with donor marrow cells
supplemented with peripheral RBCs from B6-+/+,
Gpi1b/Gpi1b,
Hbbs/Hbbs female mice. Second,
W/Wv mice were used as recipients, because they
survive long term without transplants but selectively expand donor
cells due to their heritable stem cell deficiency.30 The
use of W/Wv recipients, because they require no
irradiation to provide sites for implantation of donor stem cells, also
provided a control for the effects of irradiation on the host
microenvironment.
Donor marrow extraction, washing, adjustment of cell concentration, and
tail vein injection of the indicated number of cells (Table 1) in a volume of 0.1 mL were
performed as described previously.30 Recipient
+/+a mice were irradiated with a total dose of 10 Gy
(BMTI) or 11.5 Gy (BMTII) by 
-rays generated from a
137Cs source at a rate of 0.155 Gy/min. The protocol for
BMT experiments I and II is summarized in Table 1.
Hbb and Gpi1 phenotypes.
The -globin haplotype was determined as previously
described.31 The percentages of s (single) and d (diffuse
major and minor) hemoglobins were quantified on a Molecular Dynamics
Densitometer (Sunnyvale, CA). RBCs retrieved from the packed pellet in
a hematocrit tube were assessed for GPI1 phenotype, as
described,32 and the concentration of each isoform was
quantified as for -globin. At several time points, the proportions
of donor and host GPI1 were determined in enriched populations of RBCs,
white blood cells, platelets, and lymphocytes.33,34
Measurements of blood parameters.
Recipient blood was removed from the retroorbital sinus in a 100-µL
microhematocrit tube at 2 weeks posttransplantation and thereafter at
monthly intervals. RBCs were counted on a Coulter Counter model ZBI
(Hialeah, FL).
Histopathology.
Heart, brain, liver, kidney, and spleen were collected from mice
perfused transcardially with 1× phosphate-buffered saline (PBS)
followed by Bouin's fixative. Body, spleen, and heart weights were
determined postfixation. Tissues were embedded in paraffin, followed by
routine staining with hematoxylin and eosin (H&E). Gomori's stain was
used to detect nonhemoglobin iron. Thrombi and emboli in heart and
brain sections were distinguished from postmortem blood clots by the
presence of fibrin filaments throughout the thrombus. Infarcted areas
in both brain and heart sections were scored by the identification of
necrotic cells within an area of tissue and by a clear boundary between
affected and unaffected areas of tissue.
Statistics.
All statistical analyses were performed using the unpaired
t-test or using the unpaired nonparametric t-test where
noted, using the Instat program.
| |
RESULTS |
GPI1 assays indicate that donor cells repopulated the +/+a
recipients.
Confirmation that the sph/sph and +/+ donor marrow cells
implanted and subsequently amplified was a prerequisite for determining the effects of hematopoietic cells on thrombus formation. In the +/+a recipients, a genetic marker (Gpi1) was used
to detect donor cells. The conversion from the host GPI1AB phenotype to
the donor GPI1BB phenotype in the RBCs was monitored over time.
Table 2 shows the number of mice in each
experimental group that were 100% donor type at 2, 6, and 10 weeks
posttransplantation. Repopulation with sph/sph RBCs lagged
noticeably behind that of +/+ RBCs in BMTI. We hypothesized that the
delay was fostered by insufficient irradiation of the recipients,
generating a situation in which the sph/sph bone marrow was
at a competitive disadvantage with surviving host cells. To address
this possibility, the radiation dose per mouse was increased from 10 to
11.5 Gy in BMTII. Conversion to 100% donor GPI1 was accomplished more
rapidly among all recipients when compared with BMTI but was still
delayed in sph/sph marrow recipients when compared with +/+
marrow recipients (Table 2). The single recipient in BMTII that was not
converted at 10 weeks posttransplantation (Table 2) was 100% GPI1BB by
15 weeks posttransplantation (not shown).
All +/+
+/+a mice in both experiments and all
sph/sph+/+a mice in
BMTII remained 100% donor type throughout the duration of the
experiment. Three of the 22 sph/sph+/+a
mice in BMTI showed a resurgence of the host GPI1AB RBCs at 15 weeks
posttransplantation. One of these three maintained the host GPI type in
the RBC population for the duration of the experiment; the other two
returned to donor GPI1BB RBCs by 22 and 30 weeks, respectively.
Unexpectedly, the GPI1BB cells in the +/++/+a but
not in the sph/sph+/+a mice from BMTII
showed 100% conversion to the SS hemoglobin phenotype of the B6 RBCs
used to ensure survival of the irradiated recipients (not shown).
Regardless of the source of progenitor cells, the control mice in BMTII
were completely repopulated with control (+/+) cells.
Peripheral blood cells in the +/+a recipients were enriched
for platelets, lymphocytes, and granulocytes at 22 and 48 weeks posttransplantation in BMTI and at 15 and 42 weeks posttransplantation in BMTII. Repopulation of donor cells was not limited to RBCs. The GPI1
haplotype was 100% donor type (BB) in all transplant recipients,
including those recipients that at the same time point were not 100%
donor type in the RBC population (not shown).
RBC counts show that donor cells implanted in the
W/W v recipients.
All W/Wv recipients had a macrocytic,
normochromic anemia with RBC counts of 7.47 ± 0.15 (standard error
of the mean, SEM) × 109 cells/mL at the inception of
the experiments. Alterations in the RBC counts were therefore used as a
measure of successful donor cell implantation.
Figure 1 depicts the RBC counts ± SEM in the W/Wv recipients at four time points
posttransplantation. Results from BMTI and II were pooled,
because the differences between values were not
statistically significant. Injection of +/+ cells resulted in
alleviation of the host anemia and near normal RBC counts
(10.30 ± 0.51 × 109) by 6 weeks
posttransplantation. Injection of sph/sph cells caused a
further decrement in RBC counts to typical sph/sph
levels (3.74 ± 0.18 × 109) by
15 weeks posttransplantation. All of the
W/Wv recipients of sph/sph
marrow became moribund by 28.5 weeks posttransplantation. RBC
counts in the +/++/+a and
sph/sph+/+a mice mimicked the
data for the W/Wv recipients and further
confirmed the expansion of donor cells (data not shown).
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| Fig 1.
Mean RBC count of W/Wv mice
implanted with sph/sph or +/+ bone marrow at 2, 6, 10, and
15 weeks posttransplantation. Results are presented as mean ± SEM.
( ) +/+W/Wv, n = 10, except n = 8 at 15 weeks. ( )
sph/sphW/Wv, n = 10, except n = 7 at 15 weeks. Arrow on the y axis indicates mean RBC
count for unmanipulated W/Wv mice.
|
|
Recipients injected with sph/sph or +/+ marrow assume
the donor morphometry.
Comparisons were made at the time of autopsy to determine whether
spleen and heart weights were dependent on the donor genotype. Mice
that became moribund (those receiving sph/sph marrow) were immediately anesthetized and perfused. Healthy mice (all recipients of
+/+ cells and 4 sph/sph+/+a mice from
BMTI) were anesthetized and perfused at 50 weeks (BMTI) and 46 weeks
(BMTII). Values were not obtained from 4 sph/sph+/+a mice from each experiment
that died unexpectedly and were unsuitable for autopsy when discovered.
Figure 2A depicts spleen weight and Fig 2B
depicts heart weight, both expressed as the percentage of total body
weight. Because the data were similar for the experimental groups in
BMTI and II regardless of the date of autopsy, results were pooled.
Neither the spleen:body weight nor the heart:body weight ratios of
+/++/+a or
+/+W/Wv mice were significantly
different from those of the +/+ or W/Wv control
mice. The spleen:body weight and heart:body weight ratios of
sph/sph+/+a and
sph/sphW/Wv mice were
significantly higher than those of +/+ or W/Wv
controls (P 
.0005 for spleen and P .0028 for
heart). The morphometry of the sph/sph marrow recipients was
similar to that typically observed in unmanipulated sph/sph
mice. There seemed to be a modulating effect of the +/+a,
but not the W/Wv, host background on the donor
sph/sph marrow-induced increase in spleen weight. This was
not true of the increase in heart weight. We conclude that the host
mice acquired morphometric changes typical of the marrow donors,
further confirming donor cell repopulation.
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| Fig 2.
(A) Mean (±SEM) spleen weight (expressed as the
percentage of body weight) of control, W/Wv,
sph/sph, and BMT recipients at the time of death. () +/+
control, n = 2. () W/Wv control, n = 2. ( ) sph/sph control, n = 17. ( )
+/++/+a, n = 9. ( )
+/+W/Wv, n = 10. ( )
sph/sph+/+a, n = 32. ( )
sph/sphW/Wv, n = 10. (B) Mean (±SEM) heart weight (expressed as the percentage of body
weight) of control, W/Wv, sph/sph, and BMT
recipients at the time of death. Bars are as defined for (A); numbers
in each group are as in (A), except for
sph/sph+/+a, in which n = 28.
|
|
Recipients of sph/sph but not of +/+ cells develop
pathology typical of hemolytic anemia.
Histological observations of
sph/sph+/+a mice showed cellular casts
and debris in the proximal convoluted tubules of the kidneys, as well
as glomerular nephritis and proliferation
(Fig 3B). Both the casts and the renal
tubular epithelium were laden with hemosiderotic iron (Fig 3E). These
observations were similar to those made in sph/sph control
mice (Fig 3A and D) and in
sph/sphW/Wv mice (not shown).
The +/++/+a (Fig 3C and F) and
+/+W/Wv (not shown) mice had apparently
normal kidneys with no iron deposition.
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| Fig 3.
Histological sections of kidney, liver, heart,
and brain from sph/sph,
sph/sph+/+a, and
+/++/+a mice. (A, D, G, J, M, and P)
WBB6F1-sph/sph. (B, E, H, K, N, and Q)
sph/sph+/+a. (C, F, I, L, O, and R)
+/++/+a. (A through C) Kidney sections
stained with H&E. (D through F) Identical kidney sections as in (A)
through (C) stained with Gomori's iron stain, which stains
nonhemoglobin iron blue (D and E). (G through I) Liver sections
stained with H&E. Clusters of extramedullary hematopoiesis (arrows)
stain purple (G and H). (J through L) Identical regions of liver as
shown in (G) through (I) stained with Gomori's iron stain. (M through
O) Heart sections stained with H&E showing the large atrioventricular
valve. Note the presence of thrombi (indicated by arrows) in the valves
of (M) and (N), whereas the valve of (O) is unobstructed. (P through R)
Sagittal brain sections stained with H&E showing the cerebellar region
of the brain. Note the degeneration (arrows) of portions of the
cerebellum in both (P), in which about one third of the cerebellum is
missing due to infarction, and (Q), in which an interior portion of the
cerebellum is infarcted. Note also the lack of degeneration in (R).
|
|
Livers in the sph/sph controls (Fig 3G), the
sph/sph+/+a mice (Fig 3H), and the
sph/sphW/Wv mice (not shown)
had sites of extramedullary hematopoiesis. Iron was present in the
hepatocytes of the liver, but not in the regions of hematopoiesis (Fig
3J and K). Recipients of +/+ marrow had histologically normal livers
(Fig 3I and L). Spleen histology (not shown) from
sph/sph+/+a and
sph/sphW/Wv mice showed an
expanded red pulp but no evidence of iron deposition, consistent with
observations previously made for sph/sph mice.20 No expansion of red pulp was noted in the spleens of
+/++/+a and +/+W/Wv
mice. The histological observations made in recipients of
sph/sph bone marrow are consistent with the pathology of a
severe hemolytic anemia.
Thrombotic and embolic lesions are present in recipients of
sph/sph cells.
Histological examination of heart sections showed the presence of
thrombotic lesions in the atria and/or atrioventricular valves
of recipients of sph/sph marrow (Fig 3N). These thrombi were
similar in appearance, although smaller than the thrombi seen in 100%
of adult sph/sph mice (Fig 3M). In contrast to the kidney,
liver, and spleen pathology noted in all recipients of sph/sph marrow, thrombotic lesions were not detected in the
heart of every sph/sph+/+a or
sph/sphW/Wv mouse
(Table 3). Thrombotic lesions were not
detected in the heart of any of the recipients of +/+ marrow (Table 3
and Fig 3O) or in unmanipulated +/+a and
W/Wv controls (not shown).
In the cerebellum of sph/sph (Fig 3P),
sph/sph+/+a (Fig 3Q), and
sph/sphW/Wv (not shown) mice,
areas of infarction were noted. Figure 3P and 3Q show clearance of
necrotic tissue, leaving an area devoid of any cellular structure; this
is typical of an insult that occurred some time previously. Infarcts,
although more frequent in the cerebellum, were also seen in the
hippocampus and surrounding brain tissue. Microthrombi in blood vessels
were occasionally detected near the areas of infarction (not shown).
Given the extent of cerebellar damage in some of these mice, it was
surprising that only two mice showed any visible signs of neuromotor
defects. Neither infarctions nor thrombi were present in brains from
+/++/+a (Fig 3R),
+/+W/Wv, or +/+a and
W/Wv control mice (not shown). The overall
incidence of brain infarctions in the
sph/sph+/+a and
sph/sphW/Wv mice is shown in
Table 3. No recipient of sph/sph marrow showed evidence of
brain infarctions without also having thrombotic lesions in the heart
(data not shown), suggesting that the brain lesions are due to emboli
emanating from the heart thrombi.
| |
DISCUSSION |
The recent discovery of thrombotic lesions in all sph/sph
mice20 provides an opportunity to assess the genesis of
thrombus formation in a genetically homogeneous animal model that can
be examined sequentially over time. In the current report, our
hypothesis is that the thrombotic events are triggered by hematopoietic
cells, not nonhematopoietic cells, that are deficient in -spectrin. To test this theory, marrow transfers between affected and normal animals were performed. The results (Table 3) indicate that the thrombi
are due to -spectrin defects in cells derived from sph/sph bone marrow. The thrombi are not due to host irradiation or to the
transplantation, because neither the +/+irradiated
+/+a nor the +/+nonirradiated
W/Wv mice are affected.
A high proportion (78%) of the sph/sph bone marrow
recipients in these experiments developed cardiac thrombi. It is
possible, given their overall smaller size, that thrombi in some mice
were either missed through selective examination of heart sections or
dislodged from the heart by the perfusion process. The lack of brain
lesions without concomitant heart thrombi in sph/sph marrow
recipients is an exciting finding not previously reported for
sph/sph mice. This suggests that the brain pathology in these mice is directly related to the presence of thrombi in the heart. The
smaller cardiac thrombi seen in the mice in our experiments were
probably less subject to the shear forces present in the heart and
therefore less likely to produce emboli that would travel to the brain.
The incidence of brain lesions (38%) is higher than the incidence of
heart thrombi (15%) in -spectrin-deficient (ja/ja) mice,20 suggesting that only some of the brain lesions
result from emboli produced from heart thrombi. Alternatively, it is possible that, in some ja/ja mice, the entire heart thrombus
has dislodged and traveled to the brain. Cardiac thrombosis has also been documented in a mouse homozygous for a knockout of the band 3 gene29 but has not been investigated in an independently
generated band 3 knockout mouse.18 Thrombosis is a major
factor contributing to morbidity and mortality in patients with SCD,
PNH, and thalassemia.1-5 The use of magnetic resonance
imaging technology has indicated that the incidence of silent
thrombotic events in patients with SCD is much higher than originally
thought.35,36 Thrombosis has also been reported in one
patient with HS.6 The low incidence of thrombosis in human
HS may reflect the fact that many patients with severe HS are
splenectomized,9 removing a major source of the hemolysis
that may trigger thrombotic events. Splenectomy is immediately lethal
in mice with severe HS, probably because the spleen serves as a major
site of murine erythropoiesis.
A possible explanation for the thrombotic lesions is that the donor
hematopoietic cells create a toxic environment required for clot
formation. All recipients of sph/sph cells have extensive pathological changes in the liver, spleen, and kidneys due to transfer
of the hemolytic anemia. Such alterations may contribute to thrombotic
events by increasing the concentration of certain serum proteins that
are indicative of thrombotic risk in humans with SCD.37-40
This fails to completely explain the thrombotic events in our mice,
because recipients of sph/sph cells that develop thrombi in
the heart do not show more extensive tissue pathology than those that
do not have heart thrombi. In addition, our previous work has shown
that the severity of hemolytic anemia does not correlate with the
prevalence of thrombotic lesions in mice with HS.20
Nevertheless, the possible contribution of hemolysis-initiated toxicity
to thrombogenesis in sph/sph mice cannot at this point be
excluded.
It is not possible to limit causation of thrombi to the
sph/sph erythroid lineage, because, at least in the
irradiated hosts injected with genetically marked donor cells, all
circulating hematopoietic cells are donor type. Donor cells other than
the erythroid population may also be deficient in -spectrin.
Alternatively, donor hematopoietic cells that do not express
-spectrin may interact with defective cells to stimulate
thrombogenesis. Recent studies have suggested that thrombogenesis in
SCD may be related to increased erythrocyte phosphatidylserine exposure
and platelet-RBC adhesion.41,42 Investigation of
phosphatidylserine exposure on RBCs from mice with HS and studies of
RBC adhesion in sph/sph mice are currently under way.
Platelets have a spectrin-based cytoskeleton, but it is not clear, due
to the nature of the antibodies used, whether it is erythroid spectrin
or nonerythroid fodrin.43 Phagocytic monocytes are not
known to express -spectrin, but are known to induce local fibrin
deposition during oxygen deprivation,44 a likely side
effect of the hypochromic, macrocytic anemia in the HS mice. The
predisposition to thrombotic lesions in sph/sph mice provides
a new model in which to assess factors that may be involved in the
thrombogenic process. It is clear from our current experiments that
sph/sph hematopoietic cells are sufficient to induce
thrombosis. Repopulation with sph/sph-derived lineages of
selected hematopoietic lineages in host (+/+) mice will allow further
definition of the sph/sph hematopoietic lineages involved in
thrombogenesis in these mice. In addition, the continuing delineation of erythroid cytoskeletal gene expression in nonerythroid hematopoietic tissues in both humans and mice will also aid in the elucidation of the
thrombotic process in hemolytic anemia.
| |
ACKNOWLEDGMENT |
The authors greatly appreciate the technical assistance of Susan Deveau
and Nancy Hamblen. We thank Drs David Harrison and David Serreze for
critical review of the manuscript. We thank the Biological Imaging and
Graphics Services at the Jackson Laboratory for photographic assistance
(partially funded by National Institutes of Health [NIH] Core Grant
No. CA34196).
| |
FOOTNOTES |
Submitted June 16, 1998;
accepted August 18, 1998.
Supported by National Institutes of Health (NIH) NRSA F32 DK09482
(N.J.W.), NIH F32 DK09054 (T.M.K.), and NIH Grant No. R01 HL29305
(J.E.B.).
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.
Address reprint requests to Nancy J. Wandersee, PhD, The Jackson
Laboratory, 600 Main St, Bar Harbor, ME 04609; e-mail:
njw{at}aretha.jax.org.
| |
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Top
Abstract
Introduction
