Clinical and Experimental Pharmacology and Physiology (2007) 34, 1272–1275
Blackwell
Original Publishing
Articles al. of Asia
EFFECT OF THE HEME OXYGENASE INDUCER HEMIN ON BLOOD
GY
Anticoagulant
Rochefort et
effect hemin assessed by US
HAEMOSTASIS MEASURED BY HIGH-FREQUENCY ULTRASOUND
Gaël Y Rochefort,* Rachel Libgot,† Nicolas Desbuards,* Deborah Schlecht,* Jean-Michel Halimi,*
Frederic Ossant,† Veronique Eder* and Daniel Antier*
*LAB.P.ART. EA3852 IFR135 and †LUSSI FRE CNRS 2448, François Rabelais University, Faculty of Medicine, Tours, France
SUMMARY
1. Heme compounds, like hemin, a heme oxygenase-1 inducer,
are used in the treatment of acute porphyria treatment. The
side-effects of hemin on haemostasis have been reported. To
address those effects, in the present study we used a sensitive,
high-frequency ultrasound technique to record acoustic velocity
and to investigate whole blood clotting in Wistar rats treated
chronically with hemin (50 mg/kg per day).
2. The hemin-induced disturbances in haemostasis measured
were comparable to the heparin reference treatment, with a sig-
nificant decrease in clotting velocity in both groups compared
with controls (e.g. the time to clot was 40 ± 5, 53 ± 13 and
10 ± 2 min, respectively; P < 0.05). Precautions must be taken
when using high doses of hemin or in the treatment of bleeding
diseases.
3. Further investigations are required to explore the effects
of hemin in thrombosis models, because it could be a promising
‘old drug’ for the treatment of venous thrombosis in patients.
Key words: haemostasis, hemin, high-frequency ultrasound,
rat.
INTRODUCTION
Heme compounds are the most efficient in the treatment of acute
porphyria,1,2 a well-defined autonomic dominant disorder of heme
biosynthesis with a genetic frequency of 1 : 10 000–1 : 50 000 and
characterized by acute life-threatening attacks of non-specific
neurological symptoms.3–6 Given intravenously at moderate doses
(3– 4 mg/kg per day), heme compounds usually result in a rapid
clinical improvement, often within 1–2 days, especially when therapy
is started early in an attack.4,7,8 Such compounds are now available as
heme arginate (Normosang®; Orphan Europe, Puteaux, France) and
as haematin (Panhematin; Ovation Pharmaceuticals, Deerfield, IL,
USA), which is a lyophilized powder of hemin. Haematin infusions
have also been reported to be a safe and useful therapeutic modality
to maintain remission of allograft erythropoietic protoporphyria and
to prevent further recurrence of protoporphyric graft disease.9
In addition to therapeutic properties, side-effects of hemin have
been identified, including haemolysis and a transient anticoagulant
Correspondence: Daniel Antier, LABPART–EA 3852 10 Bd Tonnellé BP
3223, 37032 Tours Cedex 1, France. Email: antier@med.univ-tours.fr
Received 30 December 2006; revision 16 April 2007; accepted 13 May 2007.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Asia Pty Ltd
doi: 10.1111/j.1440-1681.2007.04720.
effect.4,7,10 Heme arginate does not affect coagulation, whereas hae-
matin has often been associated with coagulopathy and occasional
bleeding.11,12 A degradation product or derivative of haematin, namely
haematin-derived anticoagulant, was found to induce prolongation
of clotting times in vitro.13 Indeed, hemin has been associated with
defects in haemostasis characterized by elevation of prothrombin
time, partial thromboplastin time and thrombin time,5 and it has been
proven to suppress haemorrhagic shock.14 Therefore, hemin was
hypothesized to exert its anticoagulant effects by inactivating
thrombin.5 Conversely, hemin has been associated with no adverse
effects on haemostasis in healthy volunteers.3
Hemin can also cause liver injury, leading to coagulation disorder.
However, You et al. performed a toxicity study in rats treated with high
doses of hemin (93, 168 and 300 mg/kg per day) and reported no
liver or renal dysfunction.15 Odaka et al. reported that 30 mmol/kg,
s.c., (i.e. 20 mg/kg) hemin treatment induced heme oxygenase-1
(HO-1) and prevented halothane-induced hepatotoxicity.16 Similarly,
Chiu et al. showed that 30 mmol/kg, i.p., hemin may be hepato-
protective in rats because HO-1 is suggested to be an important com-
ponent of the anti-oxidant defence during hepatotoxicity.17 Finally,
Morio et al. reported that hemin treatment with a dose of 50 mmol/kg
(i.e. 35 mg/kg) had minimal effects on the liver, suggesting that HO-1
may play a limited role in hepatotoxicity.18
Clinical assessment of the blood clotting mechanism is usually
made by measuring the time necessary for a plasma sample to clot.
Coagulation, clot formation and thrombosis are already assessed
using non-invasive processes, such as ultrasound techniques.19–21
During the clotting process, specific changes in several ultrasound
parameters, such as velocity, attenuation coefficient and backscatter
coefficient, can be recorded quantitatively.22–24 Thus, high-frequency
ultrasound techniques have been evaluated and compared with
reference methods (i.e. the manual tilt tube and the fibrometer) and
are validated for clinical use, allowing a highly sensitive description
of the clotting process.20,25
The aims of the present study were to determine the anticoagulant
effect of hemin using in vitro high-frequency ultrasound analysis of
whole blood clotting and to evaluate the haemostatic effect of a high
dose of hemin (50 mg/kg per day).
METHODS
Experiments were performed in accordance with the Guidelines for the Care
and Use of Laboratory Animals published by the US National Institutes of
Health (NIH Publication no. 85–23, revised 1996; http://grants1.nih.gov/
grants/olaw/GuideBook.pdf ), with European Directives (86/609/CEE; http://
eur-lex.europa.eu/LexUriServ/site/en/com/2001/com2001_0703en01.pdf ),
in accordance with the regulations of the official edict of the French Ministry
of Agriculture and approved by the local ethics committee.
 Anticoagulant effect of hemin assessed by US 1273

Animals, treatment and sampling Statistical analysis

Male Wistar rats (approximately 350 g; Janvier, Le Genest-saint-Isles, France)
were chronically injected with 50 mg/kg per day, i.p., hemin in 0.2 mL
dimethylsulphoxide (DMSO; both from Sigma, Saint-Quentin Fallavier,
France; n = 5) for 7 days. Untreated (control; n = 6) and heparin-treated
(500 IU/kg per day; n = 5) rats were used as a reference. After the 7 day treat-
ment and 2 h after the last injection, whole blood samples were collected
into 3.5 mL vacuum citrated tubes (3.2%) and heated at 37.0 ± 0.1∞C. The
coagulation process was initiated by the addition of calcium chloride (100 mL
of a 0.5 mol/L solution) and acoustic measurements were recorded in a blood
chamber within 30 min after sampling, in an incubator adjusted at 37.0 ±
0.1∞C, as described previously.20,21
High-frequency ultrasound analysis
A 45 MHz focused transducer, with a broadband (200 MHz) pulser/receiver
(both designed by Panametrics, La Garenne Colombes, France), was set on
a measurement cell in order to adjust the focal depth of the transducer to
the middle of the blood chamber propagation path. The technique used to
measure acoustic velocity (m/s), and described elsewhere,20,21 was a classical
differential pulse echo method. Briefly, the time interval between ultrasound
pulses from the top and bottom of the blood chamber was recorded. Vari-
ations in the interval time were due to changes in the velocity in the blood
sample and allowed estimation of the blood acoustic velocity of the sample.
All data are expressed as the mean±SEM. Differences between control and
hemin-treated groups were analysed with the Mann–Whitney test. Overall
statistical significance was set at P < 0.05.
RESULTS
Serum heme levels were either undetectable or < 1 mmol/L in all rats
before treatment, but serum heme levels increased significantly
(n = 5; P < 0.005) to 59.6 ± 9.9 mmol/L after 1 week of daily treat-
ment with 50 mg/kg per day hemin.
Mean acoustic velocities as a function of time measured for
untreated controls, heparin-treated (reference) and 50 mg/kg per day
hemin-treated rats were recorded and the results are shown in Fig. 1.
Indeed, the clotting process for 50 mg/kg per day hemin-treated rats
is comparable to that seen in heparin-treated animals.
Mean values for time t0, slope S and the final value I for the three
groups are shown in Fig. 2. Whole blood from control rats began to

Recorded parameters
Acoustic velocities were digitized at 500 MHz with 8-bit resolution, recorded
with a 15 s time resolution and expressed as a function of the experiment
time.
For each blood samples, three characteristics were extracted: (i) the time,
t0, from which the acoustic velocity increased strongly, which corresponds
to serum expulsion and the beginning of blood clotting;20,21 (ii) the slope, S,
of the increase in acoustic velocity, which is related to the transition from
liquid to gel; and (iii) the final value, I, of the velocity at t = 100 min, which
is an index of clot density.

Measurement of serum hemin concentrations

To obtain an indirect measure of hemin metabolism after daily treatment, Fig. 1 Whole blood acoustic velocity during the clotting process. Mean
serum concentrations of heme, which is one of the metabolic products of (±SEM) acoustic velocities (in m/s) as a function of time (in min) measured
hemin, were determined, as described previously,26 prior to drug injection for untreated controls (), 500 IU/kg per day heparin ()- and 50 mg/kg
and at the end of the study. per day hemin ()-treated rats.

Fig. 2 Whole blood ultrasound parameters during the clotting process. Mean values for (a) time t0 (in min), (b) slope S (in m/s2) and (c) final value I (in
m/s) for untreated controls (), 500 IU/kg per day heparin ()- and 50 mg/kg per day hemin ( )-treated rats. *P < 0.05 versus untreated controls.

© 2007 The Authors

Journal compilation © 2007 Blackwell Publishing Asia Pty Ltd
1274 GY Rochefort et al.
clot early (low t0), rapidly (high S ) and completely (high I), whereas
whole blood from heparin- and 50 mg/kg per day hemin-treated rats
showed significant (P < 0.05) increases in t0 and significant
decreases in S and I.
DISCUSSION
After 1 week of daily treatment with 50 mg/kg per day hemin, serum
heme levels were significantly increased compared with concentra-
tions before treatment, confirming that the i.p. hemin treatment was
efficient and reproducible.
We used a highly sensitive ultrasound technique to record the
blood clotting process of animals treated or not with 50 mg/kg per
day, i.p. hemin. Intraperitoneal injections were used to avoid general
anaesthesia and bolus effects, which are often observed after i.v.
injections. All parameters recorded indicated that hemin, adminis-
tered at a high dose, causes disturbances in haemostasis, as does
heparin. Thus, the discrepancies reported in the literature3,5,11 could
be explained by individual fluctuations or clinical history. Further-
more, lower doses of hemin could induce limited haemostatic effects,
as suggest previously by Maeshima et al.14 regarding the ability of heme
arginate to induce HO-1, the rate-limiting enzyme in heme catabo-
lism involved in the inhibition of platelet-dependent thrombosis.27
Heme oxygenase-1 is ubiquitously distributed and strongly induced
by oxidative, nitrosative, osmotic and haemodynamic stresses.28
Induction of HO-1 results in the catabolism of the pro-oxidant heme
to bile pigments, biliverdin and bilirubin, which are potent anti-
oxidants capable of scavenging peroxy radicals and inhibiting lipid
peroxidation, providing an important cellular defence mechanism
against tissue injury.29 Moreover, HO-1 is responsible for the liber-
ation of the diatomic gas CO, which has significant cytoprotective
effects, including anti-inflammatory,29–32 antiproliferative,33,34 anti-
apoptotic35,36 and anticoagulating activity.37
Dimethylsulphoxide, the vehicle used in the present study for
hemin, has been reported to be a modulator of procoagulant and
fibrinolytic activities, as well as metastatic ability of B16 melanoma
cells when used at a concentration of 0.2% in vitro.38 Moreover,
experimental data have shown that 5% DMSO decreases the
adhesiveness of blood platelets in vitro and that DMSO may be a
good antagonist against platelet aggregation, as well as thrombus
formation, in vivo.39 These effects of DMSO were likely to be
involved in its protective effect against ischaemic injury40,41 when
used at high doses (1–30%). In the present study, the final concen-
tration of DMSO injected into rats was low (approximately 0.05%,
v/v). Moreover, DMSO has no effect on the inhibition of thrombus
formation in vivo in mice.42
In conclusion, hemin is known to be effective and well tolerated
in the treatment of acute porphyria. High doses of hemin could cause
disturbances in haemostasis, thus bleeding diseases and surgery
should be relative contraindications to the use of hemin, especially
in the case of concomitant heparin, and thus precautions must be
taken when using high doses of hemin. Finally, our results focus on
major potential perspectives for hemin in the treatment of thrombo-
embolic diseases.
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© 2007 The Authors

Journal compilation © 2007 Blackwell Publishing Asia Pty Ltd