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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1231-1236
A Histomorphometric Evaluation of Heparin-Induced Bone Loss After
Discontinuation of Heparin Treatment in Rats
By
Stephen G. Shaughnessy,
Jack Hirsh,
Mohit Bhandari,
Jeffrey M. Muir,
Edward Young, and
Jeffrey I. Weitz
From the Departments of Pathology and Medicine, McMaster University
and the Hamilton Civic Hospitals Research Centre, Hamilton, Ontario,
Canada.
 |
ABSTRACT |
Although it is well established that long-term heparin therapy
causes osteoporosis, it is unknown whether heparin-induced bone loss is
reversible when heparin treatment is stopped. To address this question,
we randomized rats to once daily subcutaneous injections of either
unfractionated heparin (1.0 U/g or 0.5 U/g) or saline for 28 days and
then followed the rats for an additional 28 days off treatment. Based
on histomorphometric analysis of the distal third of the right femur
proximal to the epiphyseal growth plate, 1.0 U/g heparin caused a 30%
loss in cancellous bone volume over the first 28 days. This was
accompanied by a 137% increase in osteoclast surface and a 60%
decrease in both osteoblast and osteoid surface. One month after
cessation of heparin treatment, no recovery in these parameters was
observed. Similarly, serum levels of alkaline phosphatase, a
biochemical marker of bone formation, which continued to decrease over
the course of heparin treatment, showed no signs of recovery in the
subsequent 28 days off treatment. To explore the mechanism responsible
for the prolonged effect of heparin on bone, we repeated the experiment giving 125I-labeled heparin in place of unlabeled heparin.
125I-labeled heparin was found to accumulate in bone during
the course of its administration, and be retained in bone for at least
56 days after stopping heparin treatment. These findings suggest that
heparin-induced osteoporosis is not rapidly reversible because heparin
is sequestered in bone for an extended period.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
OSTEOPOROSIS IS A well-recognized
complication of long-term heparin therapy.1-10 Although
only 2% to 3% of patients receiving protracted heparin therapy
develop symptomatic fractures, up to one third have subclinical
reductions in bone density.5,8,9 It is unknown whether the
effects of heparin on bone are reversible when treatment is
discontinued. This is an important question because if irreversible,
heparin could lower peak bone mass, thereby contributing to
osteoporosis later in life.
In previous studies, we used a rat model to examine the in vivo effects
of heparin on bone.11,12 Histomorphometric analysis of the
distal third of femurs from heparin-treated animals demonstrated a
significant loss of cancellous bone accompanied by increased numbers of
osteoclasts, and decreased numbers of osteoblasts, lining the
trabecular bone surface. Biochemical markers of bone turnover supported
these findings suggesting that heparin causes bone loss not only by
increasing osteoclastic bone resorption, but also by decreasing
osteoblastic bone formation.11,12
In the present study, we used the same rat model of heparin-induced
osteoporosis to determine whether the effects of heparin on cancellous
bone are reversible when heparin treatment is stopped. Thus, we treated
rats on a daily basis with pharmacologically relevant doses of heparin
or saline for 28 days. On day 28, half of the rats were killed and
their femurs subjected to histomorphometric analysis. The remaining
rats were kept off treatment for an additional 28 days before
subjecting their femurs to histomorphometric analysis. Herein we report
that the effects of heparin on cancellous bone loss are not reversible
within the 28-day period after treatment is stopped. To explore the
mechanism responsible for the prolonged effect of heparin on bone, the
experiment was repeated using 125I-labeled heparin in place
of unlabeled heparin. 125I-labeled heparin accumulated in
bone during the course of its administration and was retained in bone
for at least 56 days after stopping treatment. This suggests that the
lack of recovery in bone volume was due to sequestration of heparin
within the bone microenvironment.
| |
MATERIALS AND METHODS |
Materials.
Specific pathogen-free female Sprague-Dawley rats, 4 to 5 months old,
and weighing 300 to 325 g were purchased from Charles River
Laboratories (St Constant, Quebec). Serum alkaline phosphatase (ALP)
was measured using an assay from Sigma Chemical Co (St Louis, MO).
Unfractionated heparin was generously provided by Rhone-Poulenc Rorer
(Montreal, Quebec) and was iodinated as described
previously.13
Experimental design.
To examine the effects of heparin on bone morphology, a total of 56 animals was studied. Rats were randomized into one of three treatment
groups, each consisting of 16 rats. Two groups were given daily
subcutaneous injections of heparin at doses of either 0.5 U/g/day or
1.0 U/g/day for a total of 28 days. The third group served as an
age-matched control, and rats were given an equivalent volume of saline
instead of heparin. On day 28, half of the rats in each treatment group
were killed with 5% isofluorane and after exsanguination, their right
femurs were removed for histologic evaluation. Remaining rats were kept
for an additional 28 days with no further treatment. Eight rats were
also killed at day 0 (body weight of 325 ± 5 gm; mean ± standard deviation [SD]) and served as baseline controls. To permit
measurements of bone apposition rates and the calculation of dynamic
parameters, all animals received two intraperitoneal injections of
fluorescent markers. The first, demeclocycline (15 mg/kg), was given 10 days before the animals were killed, while the second, calcein (8 mg/kg), was given 3 days before the animals were killed.
In a second study that also included 56 rats, we examined the retention
of I125-labeled heparin in bone as a function of time.
Briefly, 56 rats (28 rats/group) were given daily subcutaneous
injections of I125-labeled heparin (0.1 U/g; 4.8 × 106 cpm/µg) mixed with unlabeled heparin (0.9 U/g) or an
equal amount of I125-labeled bovine serum albumin (BSA; 5.0 × 106 cpm/µg) as a control, for a period of 28 days. At various intervals over the first 28 days and for the next 56 days, four rats from each group were killed and the amount of
I125-heparin or I125-BSA retained in the right
femur determined by counting the entire bone in a  -counter
(Clinigamma, model 1272; Fisher Scientific, Nepean, Ontario, Canada).
Bone histomorphometry.
Bone histomorphometry was performed as described
previously.11,12 Briefly, the undecalcified distal third of
the right femur of each rat was embedded in glycolmethacrylate (JB-4
embedding medium; Analychem, Toronto, Ontario, Canada). Histologic
sections (6 to 8 µm) were obtained using a Riechert Jung microtome
(model K4), mounted, and then stained with either 1% toluidine blue or hematoxylin and eosin (H&E) before being subjected to morphometric analysis. In each case, a region 800 µm below the epiphyseal growth plate that included the entire metaphysis was subjected to light microscopy using a Merz grid.14 Sections examined in this
fashion encompassed a total tissue area of approximately 10 to 15 mm2. The following parameters were determined: (1)
cancellous bone volume, (2) osteoblast surface, (3) osteoid surface,
and (4) osteoclast surface. For each section, cancellous bone volume
was calculated from a total of > 1,600 point measurements (45 fields;
400× magnification), which were selected at random using the Merz
grid. The percent osteoblast, osteoid or osteoclast surface was
calculated under oil immersion (1,000×) by recording the presence
or absence of each where the hemispherical grid of the Merz radicule
crossed cancellous bone. Osteoblasts were identified morphologically as distinct cuboidal-shaped cells lining the cancellous bone surface, whereas osteoclasts were identified as large multinucleated cells that
stained with tartrate-resistant acid phosphatase (Sigma Chemical Co;
Procedure No. 386) and were located close to the cancellous bone
surface. The histomorphometric parameters of trabecular width, number, and separation were measured directly using an epifluorescent microscope (Leica Laborlux; Willowdale, Ontario, Canada) coupled to an
IBM computer (Hewlett Packard, Toronto, Ontario, Canada). Images were
captured using a CCD video camera module electronically linked to a
computer imaging software system (Northern Exposure; Empix Inc,
Mississauga, Ontario, Canada). Measurements of erosion depth were
also determined from captured images by random measurement of the depth
of resorption lacunae that were associated with tartrate resistant acid
phosphatase-positive cells. All histological analyses were done by a
single investigator who was blinded to treatment allocation.
To quantify bone mineralization, cancellous bone surface intersecting
with the grid of the Merz radicule was scored as either labeled or
unlabeled depending on whether or not the cancellous bone surface was
fluorescently labeled. Fluorescent bone surface was further
characterized as having either single or double label according to the
number of distinct lines observed on the labeled surface.
Double-labeled perimeter was then used to calculate the dynamic
variables of mineral apposition rate and bone formation rate
(surface-based) according to the standard nomenclature described by Jee
et al15 and Parfitt et al.16
Statistical analysis.
Analysis of variance was used to compare the results in the
experimental groups with those in the controls. The significance of
differences was determined using an unpaired Student's t-test with a Bonferroni correction for multiple comparisons. All data are
expressed as a mean ± standard error of mean (SEM).
| |
RESULTS |
Effect of heparin on body weight and alkaline phosphatase levels.
During the course of the study, both heparin-treated and control rats
gained similar amounts of weight (data not shown). A time-dependent
decrease in serum ALP levels was observed in rats treated with either
0.5 U/g/day or 1.0 U/g/day heparin (Fig 1). Serum ALP levels decreased during heparin treatment and remained decreased 28 days after stopping treatment. Thus, at day 56, there was
a 44.3% ± 5.1% (P < .001) and a 33.2% ± 7.0%
(P < .001) reduction in ALP in rats given 1.0 U/g/day and 0.5 U/g/day heparin, respectively.
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| Fig 1.
Serum ALP levels both during and after the
discontinuation of heparin treatment. Rats were injected for 28 days
with vehicle alone ( ) or unfractionated heparin at concentrations of
either 0.5 U/g/day ( ) or 1.0 U/g/day ( ) and then allowed to live
for an additional 28 days with no further treatment. During the course
of this experiment, tail vein blood samples were collected weekly and
assayed for ALP activity as an index of bone formation.
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Reversibility of heparin-induced cancellous bone loss.
To examine the reversibility of heparin-induced bone loss, we compared
the extent of bone loss in the undecalcified right femur of rats that
had been treated with heparin for 28 days with that in rats that had a
28-day recovery period after a 28-day course of heparin treatment. A
region 800 µm below the epiphyseal growth plate that included the
entire metaphysis was analyzed for cancellous bone. As shown in
Fig 2, no significant difference in
cancellous bone volume (BV/TV) was found between baseline and age-matched control rats. In contrast, heparin, at doses of either 0.5 or 1.0 U/g/day, produced a significant reduction (P < .001) in cancellous bone by day 28. This remained unchanged 28 days after
stopping heparin treatment. Thus, after 28 days treatment with 1.0 U/g
heparin, there was a 30.0% ± 4.5% reduction in cancellous bone
volume when compared with age-matched controls. Twenty-eight days after
stopping heparin treatment, cancellous bone volume was still decreased
by 39.0% ± 4.1% as compared with age-matched controls.
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| Fig 2.
The reversibility of heparin-induced cancellous bone
loss. Rats were injected with vehicle alone (open bars) or
unfractionated heparin at a concentration of 0.5 U/g/day
(cross-hatched; rising to the right) or 1.0 U/g/day (cross-hatched;
rising to the left). On day 28, half of the rats from each group were
killed while the other half were allowed to live for an additional 28 days with no further treatment before determining the total epiphyseal
area occupied by cancellous bone. Data are expressed as mean ± SEM.
*P < .005 when compared with either baseline or control
values.
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Similar results were obtained when the parameters of trabecular width,
number, and separation were evaluated
(Table 1). Thus, heparin significantly
decreased both trabecular number and width when compared with either
age-matched controls or baseline values. Age-matched controls had a
mean of 9.8 trabeculae/mm2 with a mean width of 104.4 µm.
Heparin treatment (1.0 U/g/day) for 28 days reduced the number of
trabeculae to 7.5 ± 0.3 trabeculae/mm2 (P < .01), with the remaining trabeculae having a mean width of 73.7 ± 3.3 µm (P < .001). This resulted in the distance
between adjacent trabeculae increasing from 261.6 ± 9.8 µm to
296.8 ± 12.4 µm, an increase of 13.5% ± 4.7% (P < .001). Similar findings were obtained 28 days after stopping heparin
treatment (Table 1).
Reversibility of surface-based data.
In previous studies,11,12 we demonstrated that heparin has
profound effects on the numbers of both osteoblasts and osteoclasts. To
determine whether these effects were reversible, we compared surface-based data from rats allowed to recover for 28 days after heparin therapy was stopped with the results obtained immediately after
28 days of heparin treatment. The parameters that were evaluated included: (1) percent osteoblast surface (Ob.S/BS), (2) percent osteoid
surface (OS/BS), and (3) percent osteoclast surface (Oc.S/BS). As shown
in Fig 3, heparin treatment caused a
decrease in both osteoblast and osteoid surface. At a heparin dose of
1.0 U/g/day, there was a 59.0% ± 6.0% (P < .001)
decrease in osteoblast surface and a 68.3% ± 12.2% (P < .001) decrease in the percentage of cancellous bone covered by osteoid.
Similar decreases in osteoblast and osteoid surfaces were found 28 days
after stopping heparin treatment (Fig 3).
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| Fig 3.
The reversibility of heparin's effects on the percentage
of cancellous bone surface length occupied by either osteoblasts or
osteoid. Rats were injected daily with vehicle alone (open bars) or
unfractionated heparin at a concentration of either 0.5 U/g/day (cross
hatched; rising to the right) or 1.0 U/g/day (cross hatched; rising to
the left). On day 28, half of the rats from each group were killed
while the other half were allowed to live for an additional 28 days
with no further treatment before determining the percentage of
cancellous bone surface lined with either osteoblasts (A) or osteoid
(B). Data are expressed as mean ± SEM. *P < .005 when
compared with either baseline or control values for both (A and B).
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Heparin treatment causes an increase in osteoclast surface (Oc.S/BS).
As illustrated in Fig 4A, heparin (1.0 U/g/day) caused a 137.3% ± 14% (P < .001) increase in
the percentage of cancellous bone covered by osteoclasts. A similar
increase in osteoclast surface was found 28 days after stopping
heparin. Erosion depth also increased by 47.9% ± 5.4% (P < .001) in rats given 1.0 U/g heparin (Fig 4B) and remained elevated
28 days after stopping heparin treatment.
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| Fig 4.
The reversibility of heparin's effects on osteoclast
surface and erosion depth. Rats were injected with vehicle alone (open
bars) or unfractionated heparin at a concentration of 0.5 U/g/day
(cross-hatched; rising to the right) or 1.0 U/g/day (cross-hatched;
rising to the left). On day 28, half of the rats from each group were
killed while the other half were allowed to live for an additional 28 days with no further treatment before determining osteoclast surface
(A) and/or erosion depth (B). Data are expressed as mean ± SEM. * P < .005 when compared with either baseline or control
values for both (A and B).
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Reversibility of the heparin effect on dynamic measurements of bone
mineralization.
Bone mineralization was measured by fluorescent labeling over a 7-day
period. As shown in Table 2, heparin had no
effect on mineral apposition rates. In contrast, heparin treatment
significantly decreased the percentage of cancellous bone covered by
double-labeled surface (MS/BS) (29.3% ± 6.1%; P < .01)
and lowered the bone formation rate (BFR/BS) from 25.9 ± 2.7 to
19.2 ± 2.2 (P < .001). There was no significant change in
these values 28 days after stopping heparin treatment (Table 2).
Retention of I125-labeled heparin in bone.
To explore the mechanism responsible for the prolonged effect of
heparin on bone, we gave rats daily injections of
I125-labeled heparin or I125-labeled BSA for 28 days. Radioactivity in the femurs was determined at various intervals
during and after the course of heparin treatment. As illustrated in
Fig 5, accumulation of radioactive heparin
in the femur during the 28-day course of treatment was significantly greater than that of radiolabeled BSA. After cessation of heparin treatment, radiolabeled heparin levels plateaued at a significantly higher level than radiolabeled BSA, which approached baseline.
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| Fig 5.
The retention in bone of 125I-labeled heparin
over time. Rats were injected with 125I-labeled heparin
( ) or 125I-labeled BSA ( ) over a 28-day period. At
various intervals over the 28 days of heparin treatment and for an
additional 56 days thereafter, four rats from each group were killed,
their femurs isolated. The amount of heparin that had accumulated in
each femur was then determined by use of a -counter. Counts were
corrected over time for the decay of 125iodine and
converted to ng of heparin or BSA/g tissue. Data are expressed as mean ± SEM.
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DISCUSSION |
In this study, we used a well-established rat model of heparin-induced
osteoporosis11,12 to determine whether the decrease in
cancellous bone produced by heparin is reversible once heparin therapy
is discontinued. Here we report no evidence of recovery in the 4 weeks
after stopping heparin treatment, and we hypothesize that this reflects
the fact that heparin accumulates in bone and is retained long after
heparin treatment is discontinued.
Heparin could reduce cancellous bone volume by either decreasing bone
growth or by influencing the rate of bone remodeling (ie, increasing
rates of bone resorption and/or decreasing rates of bone
formation). In this study, we used mature 4- to 5-month old rats to
minimize potential effects of heparin on growth. Previously, we used
2-month old rats, which made it difficult to determine whether the
heparin-induced reduction in cancellous bone was caused by an effect on
bone growth or by effects on bone remodeling. In this study, rats given
1.0 U/g/day heparin for 28 days demonstrated a 30.0% ± 4.5%
reduction in cancellous bone; a value comparable to the 31.9% ± 3.2% reduction that we previously reported.12 This
suggests that heparin causes cancellous bone loss by influencing bone
remodeling rather than growth.
There is evidence in humans that heparin-induced reduction in bone
density is not rapidly reversible. For example, 5 of 14 women given
long-term heparin during pregnancy had a 10% or greater decrease in
femoral bone density, and none showed a significant increase 6 months
later.9 Similar results were reported in a second
study.8 A more recent case-control study compared spinal
and radial bone mineral densities in 61 premenopausal women treated
with long-term heparin therapy with those in age-matched controls.
Although there were no significant differences in mean radial and
spinal bone densities, a significantly greater proportion of women who
had received heparin 2 years previously had bone densities below a
predefined minimal level.17 Sequestration of heparin in
bone provides a plausible explanation for these results.
Spinal fractures have been reported in patients treated with long-term
( 2 months) subcutaneous heparin at doses as low as 10,000 IU/day.7,10 Doses as high as 50,000 IU/day have been used
when treating pregnant women with venous
thromboembolism.7-9 In the current study, we used dosages
of 0.5 and 1.0 U/g/day, which are equivalent in a 70 kg patient to
doses of 35,000 and 70,000 IU/day. Thus, while our dose of 0.5 U/g/day
is within the range used for prophylaxis and treatment, our dose of 1.0 U/g/day is higher than that which is normally recommended therapeutically.
The site of heparin sequestration in bone is unknown. Heparin has been
reported to bind to endothelial cells and macrophages, as well as to a
variety of plasma proteins.18-21 Such binding explains heparin's poor bioavailability at low doses and the variable
anticoagulant response that it produces when used
therapeutically.20-22 Moreover, the phenomenon of heparin
resistance may also be directly attributable to nonspecific binding of
heparin by plasma proteins.23 Our findings suggest that
heparin binds to bone marrow cells or to the cancellous bone surface.
Thus, we compared the accumulation of 125I-labeled heparin
in rat femurs with that of 125I-labeled BSA and found that
only heparin accumulated to significant levels in bone where it
remained at relatively high levels once treatment was discontinued.
Were this radioactivity a result of catabolized 125iodine,
we would expect that radioactivity levels in the
125I-labeled BSA group would also be elevated. As this was
not the case, we conclude that radioactivity in the femurs of
heparin-treated animals is likely a result of heparin sequestering in
the bone microenvironment. The sequestration of heparin within the bone microenvironment may explain the lack of recovery of bone loss over the
28-day study period after heparin therapy was stopped.
The choice of a 28-day recovery period after heparin treatment should
have been sufficient to observe an improvement in cancellous bone
volume after stopping heparin treatment. Both corticosteroid-induced bone loss24-26 and bone loss due to limb
immobilization27,28 are, at least to some extent,
reversible within a similar time frame. For example, Tuukkanen et
al27 reported that rat femur mineral mass recovered 40%
within the first 3 weeks of remobilization after 3 weeks of cast
immobilization. In contrast, we found no evidence of recovery within
the first 28 days after stopping heparin treatment. Moreover, heparin
reduces cancellous bone volume not only by reducing the mean width of
individual trabeculae, but also by reducing the number of trabeculae.
This suggests that even if there is delayed recovery in cancellous bone
volume, the distorted bone architecture would compromise bone strength.
If our findings in rats can be translated to humans, long-term heparin therapy may contribute to osteoporosis later in life. This may be
particularly important in women, given the significant bone loss that
occurs in the postmenopausal period.
| |
FOOTNOTES |
Submitted March 4, 1998; accepted October 5, 1998.
Supported by the Heart and Stroke Foundation of Ontario. J.I.W. is a
Career Investigator of the Heart and Stroke Foundation of Ontario.
Partial salary support for J.M.M. was obtained from the Canadian
Memorial Chiropractic College.
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 Stephen G. Shaughnessy, PhD, Hamilton Civic
Hospitals Research Centre, 711 Concession St, Hamilton, Ontario, Canada
L8V 1C3.
| |
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S. M. Bates, I. A. Greer, I. Pabinger, S. Sofaer, and J. Hirsh
Venous Thromboembolism, Thrombophilia, Antithrombotic Therapy, and Pregnancy: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition)
Chest,
June 1, 2008;
133(6_suppl):
844S - 886S.
[Abstract]
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R. Rajgopal, M. Butcher, J. I. Weitz, and S. G. Shaughnessy
Heparin Synergistically Enhances Interleukin-11 Signaling through Up-regulation of the MAPK Pathway
J. Biol. Chem.,
July 28, 2006;
281(30):
20780 - 20787.
[Abstract]
[Full Text]
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G. F. Pineo and R. D. Hull
Low-Molecular-Weight Heparin for the Treatment of Venous Thromboembolism in the Elderly
Clinical and Applied Thrombosis/Hemostasis,
January 1, 2005;
11(1):
15 - 23.
[Abstract]
[PDF]
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M. A. Smythe, W. E. Dager, and N. M. Patel
Managing Complications of Anticoagulant Therapy
Journal of Pharmacy Practice,
October 1, 2004;
17(5):
327 - 346.
[Abstract]
[PDF]
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S. M. Bates, I. A. Greer, J. Hirsh, and J. S. Ginsberg
Use of Antithrombotic Agents During Pregnancy: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy
Chest,
September 1, 2004;
126(3_suppl):
627S - 644S.
[Abstract]
[Full Text]
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S. M. Bates and J. S. Ginsberg
How we manage venous thromboembolism during pregnancy
Blood,
November 15, 2002;
100(10):
3470 - 3478.
[Abstract]
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G Ruiz-Irastorza, M A Khamashta, C Nelson-Piercy, and G R. Hughes
Lupus pregnancy: is heparin a risk factor for osteoporosis?
Lupus,
September 1, 2001;
10(9):
597 - 600.
[Abstract]
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J. Hirsh, T. E. Warkentin, S. G. Shaughnessy, S. S. Anand, J. L. Halperin, R. Raschke, C. Granger, E. M. Ohman, and J. E. Dalen
Heparin and Low-Molecular-Weight Heparin Mechanisms of Action, Pharmacokinetics, Dosing, Monitoring, Efficacy, and Safety
Chest,
January 1, 2001;
119(1_suppl):
64S - 94S.
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J. S. Ginsberg, I. Greer, and J. Hirsh
Use of Antithrombotic Agents During Pregnancy
Chest,
January 1, 2001;
119(1_suppl):
122S - 131S.
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Top
Abstract
Introduction