Antithrombin and Preeclampsia

Antithrombin and Preeclampsia

2 Treatment of Preeclampsia

TABLE OF CONTENTS

Antithrombin and Preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Antithrombin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Anticoagulant Properties of AT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Anti-inflammatory Properties of AT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Summary of AT Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Biochemical and Physiological Changes Seen in Preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Treatments for Preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Animal Models for Preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Use of Antithrombin in Animal Models for Preeclampsia or Other Relevant Disorders . . . . . . . . . . . . . . . . . . . . . . .7

In Vitro Studies Related to AT as an Anti-inflammatory Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Conclusions Regarding Non-clinical Studies Supporting the Use of AT for Treatment of Preeclampsia . . . . . .12

Antithrombin Replacement in Human Preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Rationale for Use of Antithrombin for Preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

FIGURES

Figure 1 Modulation of Coagulation (Roemisch et al 2002) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Figure 2 Anti-inflammatory Properties of AT (Roemisch et al 2002) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Figure 3 Interactions of AT with the Endothelium (Antithrombin Disease Monograph) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Figure 4 Anticoagulant and Anti-inflammatory Properties of AT (Antithrombin Disease Monograph) . . . . . . . . . . . . . . . . .4

Figure 5 Spiral Artery Remodeling in Preeclampsia and Normal Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Figure 6 Experimental Design (Shinyama et al 1996a) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

Figure 7 Changes in Systolic Blood Pressure (Shinyama et al 1996a) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

Figure 8 Changes in Proteinuria (Shinyama et al 1996a) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

Figure 9 Experimental Design (Shinyama et al 1996b) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Figure 10 Fibrin Deposition in Kidney, Liver and Lung (Shinyama et al 1996b) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

Figure 11 Urinary Concentration of Protein (Shinyama et al 1996b) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Figure 12 Changes in Blood Pressure (Shinyama et al 1996b) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Figure 13 Protocol for AT Use in Preeclampsia (Mangione & Giarratano 2002) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

TABLES

Table 1 Rationale for Use of AT in Preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14


A N T I T H R O M B I N A N D P R E E C L A M P S I A

To understand the possible role of antithrombin (AT) in

preeclampsia, it is important to understand (1) the pleiotropic

effects of AT on the coagulation and inflammatory pathways

and (2) the bases for the various manifestation of preeclampsia

in pregnancy .

AntithrombinAntithrombin is a complex glycoprotein with multiple

pharmacologically important activities in both the coagulation

and inflammatory cascades . Normal serum AT levels range

between 12 .5 – 15 mg/dL (Murano et al 1980) . It is the most

critical modulator of coagulation (Figure 1) and has potent anti-

inflammatory properties (Figure 2) independent of its effects on

coagulation (reviewed in Roemisch et al 2002) .

Anticoagulant Properties of AT

AT is a serine protease inhibitor (Figure 1) that is the principal

inhibitor of the blood coagulation serine proteases thrombin

and Factor Xa, and to a lesser extent, Factors IXa, XIa, XIIa, trypsin,

plasmin, and kallikrein (Aubry & Bieth 1977, Lahiri et al 1976,

Menache 1991, Menache et al 1992, Travis & Salvensen 1983) .

Binding of heparin to AT results in a conformational change that

greatly increases the activity of AT toward thrombin (1000 fold)

and other serine proteases . AT neutralizes the activity of thrombin

as well as other serine proteases by forming a 1:1 stoichiometric

complex between enzyme and inhibitor (Bauer & Rosenberg

1991) . In the case of thrombin inhibition, the complex formed is

thrombin-antithrombin (TAT), which is rapidly removed from the

circulatory compartment (t1/2

= 5 min) . Complex formation occurs

at a relatively slow rate in the absence of heparin . When heparin is

present, however, it binds to lysyl residues on AT and dramatically

accelerates the rate of complex formation (Bauer & Rosenberg

1991) . The localization of a fraction of the AT bound to heparan

sulfate proteoglycans (HSPG) on the endothelial surface, where

enzymes of the intrinsic coagulation cascade are commonly

generated, enables AT to rapidly neutralize these hemostatic

enzymes and protect natural surfaces against thrombus formation

(Rosenberg 1989) .

MǾ

AT

STIMULUS LPS

INFLAMMATORYMEDIATORS

THROMBIN

EXPRESSION RELEASE

COAGULATION

TAT COMPLEX

GAGS

ENDOTHELIUM

CLEARANCE

PROSTACYCLIN

ADHESION

THROMBIN

THROMBIN

TF

PLATELETS

AGGREGATION

FIGURE 1 Modulation of Coagulation (Adapted from Roemisch et al 2002)

FIGURE 2 Anti-inflammatory Properties of AT (Adapted from Roemisch et al 2002)

Anti-inflammatory Properties of AT

Antithrombin has been shown to have significant anti-

inflammatory properties . AT binds to heparin-like

glucosaminoglycans on the surface of endothelial cells in vitro

(Yamauchi et al 1989, Horie et al 1990) and in vivo (Harada

et al 1999) . This binding promotes endothelial cell release

of prostacyclin (PGI2), which, in turn, impacts a variety of

inflammatory processes .

It has been demonstrated that both endotoxin-induced

pulmonary vascular injury (Uchiba et al 1996) and ischemia/

reperfusion-induced liver injury (Harada et al 1999) were

reduced by AT treatment through its promotion of endothelial

PGI2 production (Figure 3) . These effects were not observed

with a Trp49-modified AT, which is incapable of promoting the

endothelial release of PGI2 due to its lack of affinity for heparin .

ENDOTHELIUM

MǾ

AT III

RESPIRATORY BURST

CAPILLARY LEAKAGE

INFLAMMATORY MEDIATORS

CY TOKINESIL-6

IL-8 IL-8

TF

ROLLING | STICKING | DIAPEDESIS

CELL STIMULATION

MEDIATORS

MEDIATORS LPS

NEUTROPHILCOAGULATION CONSUMPTION

LYSOSOMAL ENZYMES

INFAMMATORY MEDIATORSINFLAMMATORYSITE


LPS

HSPG

LPS

BLOCK

HSPG

PGI2

HSP6

Endothelium

Thrombin

Thrombin

Heparin

AT

AT AT

AT

AT

IL-6IL-6

IL-6IL-6

FIGURE 3 Interactions of AT with the Endothelium (Data on file, rEVO Biologics, Inc.)

FIGURE 4 Anticoagulant and Anti-inflammatory Properties of AT (Data on file, rEVO Biologics, Inc.)

Ostrovsky et al (1997), using a cat mesentery ischemia-reperfusion

injury model, showed that AT inhibits leukocyte rolling and

adhesion that are characteristic of inflammatory reactions . This

effect was independently confirmed by Hoffman et al (2000) and

Nevière et al (2001) using intravital videomicroscopy to assess small

intestine injury in endotoxemic rats treated with AT . In both cases,

co-treatment with indomethacin, an inhibitor of PGI2 production,

abolished the effect of AT on leukocytes . Specific interaction with

cell-surface glycosaminoglycans on the endothelium is also the

mechanism of antithrombin-mediated attenuation of leukocyte-

endothelial cell interaction Hoffman et al (2002) .

Examining the intracellular mechanism of AT anti-inflammatory

activity, Oeschlager et al (2002) used cultured human monocytes

and endothelial cells to show that AT inhibits NF-κB induction .

Since the NF-κB transcription factor activates genes that

encode defense and signaling molecules in response to various

inflammatory stimuli (Hatata et al 2000), inhibition of this

signaling pathway represents a plausible mechanism for AT’s

anti-inflammatory effects .

Summary of AT Properties

In summary, it is clear that AT, in addition to its central role in the

coagulation cascade, has potent anti-inflammatory activities

(Figure 4) . Since the anti-inflammatory properties of AT are

mediated through its interaction with heparin-like receptors such

as syndecan-4, located on endothelial surfaces and leukocytes, supra-

physiological doses are necessary to achieve a significant effect .

BlockAT

IL-6

TFPI

Factor Xa

Coagulation

ICAM-1F-Selectin

IL-6, IL-8

Thrombin

Fibrin Fibrinogen

Neutrophilactivation rollingand infiltration

Coagulation

IL-6, PAF, P-Selectin

PGI2

Release

Endothelium

Site of Coagulation/Inflammation

Thrombin AT

Bloc

k

BlockBlock

Block Promote

PreeclampsiaHigh blood pressure is one of the most common complications

in pregnancy and includes both preeclampsia and eclampsia

(reviewed in Onisai et al 2009) . Preeclampsia is a disorder of

pregnancy developing after 20 weeks of gestation in 5-8% of

pregnant women worldwide, which can cause maternal and fetal

morbidity and mortality . The disorder appears to be an aberrant

interaction of the placenta of the fetus with the cardiovascular

and immune systems of the mother (James et al 2010) . As a result,

the mother develops hypertension and proteinuria and may

develop disseminated intravascular coagulation (DIC), acute renal

failure, stroke (ischemic, due to vasospasm and microthrombosis

or even hemorrhagic due to severe thrombocytopenia), acute

pulmonary edema, cerebral edema, placental abruption, liver

hemorrhage/ rupture, transformation into chronic hypertension,

or even maternal death (preeclampsia is the second most

frequent cause of maternal death linked to pregnancy) . As a

result of the placental insufficiency, the fetus may also be affected

resulting in: pregnancy loss, fetal death in utero, intrauterine

growth restriction (IUGR) and premature labor (Onisai et al 2009) .

The causes of preeclampsia still remain somewhat elusive

but are believed to be multifactorial . Preeclampsia has been

described as a 2-stage disorder (reviewed in James et al 2010

and Grill et al 2009), starting with defective implantation in the

1st trimester, followed by placental-induced clinical morbidity

in the 3rd trimester . Preeclampsia involves maternal gestational

hypertension (systolic and diastolic blood pressure of ≥ 140

and 90 mm Hg, respectively, on two occasions, at least 6 hours


Nature Reviews | Immunology

No pregnancy Pre-eclampsia Normal pregnancy

Myometrium

Decidua

Decidua–myometriumboundary

Placenta

Arcuateartery

Trophoblast

Spiral artery

FIGURE 5 Spiral Artery Remodeling in Preeclampsia and Normal Pregnancy (Used by permission)

apart), proteinuria (protein excretion of ≥ 300 mg in a 24 h

urine collection, or a dipstick of ≥ 2+) (Grill et al 2009), systemic

endothelial cell activation and an exaggerated inflammatory

response . Research has shown that the presence of the placenta

is required for the disorder and that the only currently effective

treatment is delivery of the infant along with the placenta . (Mutter

and Karumachi 2008) .

In the asymptomatic Stage 1 of preeclampsia in the 1st trimester,

the first pathophysiological occurrence is inadequate placentation

characterized by incomplete decidual spiral artery remodeling

(Figure 5) (reviewed in LaMarca et al 2008; Mutter and Karumachi

2008; Onisai et al 2009 and James et al 2010) . The spiral miometrial

arteries are not correctly invaded by the fetal trophoblast and

the vessels remain as small vessels, capable of vasospasm and

increased vascular reactivity . Additionally, there is failure of the

cytotrophoblasts to convert from a more epithelial to endothelial

phenotype (Hladunewich et al 2007) . Placental ischemia occurs,

followed by the release of a number of vasoactive factors

(increased release of thromboxane A2 and endothelin-1 - both

highly vasoconstrictive and decreased synthesis of nitric oxide

(NO) and prostacyclin- both natural vasodilators) . Chronic hypoxia

or alternate periods of hypoxia/re-oxygenation within the

intervillous space triggers tissue oxidative stress and increased

placental apoptosis and necrosis .

In Stage 2, the clinical disorder arises when the maternal vascular

and immune systems can no longer handle the increased

shedding of placentally-produced debris (syncytiotrophoblast

knots and microparticles) and the aberrant expression of pro-

inflammatory, anti-angiogenic and angiogenic factors, leading

to systemic endothelial cell and platelet dysfunction and an

exaggerated inflammatory response (Grill et al 2009 and James

et al 2010) . In a positive feedback loop, the endothelial and

platelet cells release active factors that stimulate and maintain

the endothelial and platelet dysfunctions (James et al 2010) .

Biochemical and Physiological Changes Seen in Preeclampsia

In preeclampsia, a variety of biochemical and physiological

changes are seen in the maternal cardiovascular and

immune systems .

As reviewed in Onisai et al (2009), the hematological changes in

the cardiovascular system that occur in a preeclampsia pregnancy

fall into 3 major categories:

1 . The first category includes the platelet anomalies, platelet

dysfunction and thrombocytopenia, which may be

potentially life-threatening . The release of thromboxane A2

and decreased production of prostacyclin described above

leads to an imbalance in thromboxane A2/prostacyclin

ratio which favors the promotion of vasospasm, induces

supplementary platelet aggregation and endothelial damage .

These events contribute to the maintenance of platelet

dysfunction and promote platelet consumption (activation,

aggregation, microangiopathic hemolysis induced by

severe vasospasm), resulting in thrombocytopenia, and an

important sign of severe/aggravating preeclampsia . Excessive

platelet activation is associated with endothelial dysfunction,

thrombosis in microcirculation, end organ degenerative

necrosis and placental infarction .

2 . The second category includes alterations in hemoglobin

and erythrocytic parameters due to increased endothelial

permeability . In some cases, there may also be anemia due to

physical destruction of erythrocytes in the microcirculation

affected by disseminated microthrombosis .

3 . The third category includes the coagulation changes .

Preeclampsia is a highly thrombotic and procoagulant

state with platelet activation and consumption, promotion

of thrombin formation and promotion of fibrin formation

and destruction . As compared to normal pregnant women,


preeclamptic pregnancies show significantly increased levels

of TAT complexes and PAI-1, while fibrinogen, antithrombin

and PAI-2 levels are significantly reduced .

As a result of the aberrant spiral artery conversion, intermittent

periods of hypoxia and reoxygenation occur in the placenta

leading to oxidative stress injuries . These injuries include

widespread placental lipid and protein oxidative modifications,

mitochondrial and endoplasmic reticulum stress and tissue

apoptosis and necrosis . The oxidative stress and endothelial

activation can also stimulate IL-6 release, which increases the

endothelial permeability and may reduce prostacyclin synthesis

by inhibiting the cyclooxygenase . The result is an increased

thromboxane A2/prostacyclin ratio, which is found

in preeclampsia .

Due to the apoptosis and necrosis in the placenta, the maternal

circulation is flooded with debris released from the placenta .

Although such debris is usually released during the progression

of a normal pregnancy, during preeclampsia, the quality and

the amount of debris is much more than normal . The placental

debris includes multinucleated syncytiotrophoblast knots,

microparticles (STMP) and nanoparticles (exosomes) . STMPs may

affect both endothelial cell and lymphocyte proliferation . In vivo,

STMPs bind to circulating monocytes and stimulate production

of the inflammatory cytokines TNFα and IL-12 and -18, with the

production of these cytokines significantly increased in STMPs

from preeclamptic patients (reviewed in James et al 2010) .

In addition to the hematological changes and the increased

release of placental debris, a number of circulating soluble factors

that contribute to endothelial dysfunction in preeclampsia

have been identified (reviewed in Mutter & Karumanchi 2008

and James et al 2010) . The circulating factors include cytokines

(TNFα, IL-6, IL-1α, IL-1β), apoptotic factors (Fasligand, oxidized

lipid products, neurokinin B and asymmetric dimethylarginine)

and anti-angiogenic factors (fms-like tyrosine kinase-1 (sFlt-

1) and endoglin) . It has been found that the hypertension

and proteinuria are due to excess circulating soluble fms-like

tyrosine kinase-1 (sFlt-1), which is produced by the placenta

and neutralizes vascular endothelial growth factor and placental

growth factor . In addition, soluble endoglin synergizes with sFlt-1

and contributes to the pathogenesis of preeclampsia .

The inflammatory process in preeclampsia involves activation

of leukocytes (possibly by SMTPs), which mediate adhesion of

neutrophils to the endothelium . Following activation and binding,

neutrophils then transmigrate through the vascular wall releasing

cytokines that mediate vasoconstriction and vascular damage,

including TNFα, IL-1 and IL-8 .

While a progressive increase in serum inflammatory profile is a

physiological feature in a normal pregnancy, the exacerbation

of this response in preeclampsia likely further contributes to

the hypertensive effects of this condition, as increasing medical

evidence suggests that hypertension-associated vascular disease

is an inflammatory process (James et al 2010) .

Treatments for Preeclampsia

In most cases of preeclampsia, safe prolongation of the pregnancy

by days or weeks is highly desirable to reduce morbidity and

mortality in the infant and mother . There is disagreement about

management of patients with severe preeclampsia prior to 34

weeks of gestation . Antihypertensive agents, diuretics, sedatives

and chronic parenteral magnesium sulfate have been used as

temporizing measures in some cases (Ferrazzani 1999) .

However, the only effective treatment for preeclampsia is delivery

of the infant and the placenta . If part of the placenta is retained

following delivery, the disorder persists . The importance of the

placenta to preeclampsia is demonstrated in women with a

molar pregnancy (placenta without a fetus) . These women also

experience preeclampsia (Mutter and Karumachi 2008) .

Animal Models for PreeclampsiaPreeclampsia only occurs naturally and spontaneously in women

and higher apes . It has been speculated that an upright posture

and uteroplacental ischemia are necessary for development of the

full spectrum of changes associated with preeclampsia in humans

(Podjarny et al 2004) . Although a variety of animal models have

been evaluated for their relevance to the study of preeclampsia,

to date, none of the models fully duplicate the events seen in

the progress of preeclampsia in pregnant women (Podjarny et

al 2004) . In fact, in their review of the current animal models for

preeclampsia, Podjarny and colleagues have concluded:


“These models are definitely of use in preeclampsia

research but because this disease only occurs

spontaneously in primates, the definitive studies on

preeclampsia will, of necessity, be clinical” .

In spite of this, parts of the multifactorial preeclampsia syndrome

have been induced in animal models, which have produced data

that may be helpful in developing treatments to ameliorate this

disorder in pregnant women .

Use of Antithrombin in Animal Models for Preeclampsia or

Other Relevant Disorders

Two studies utilizing antithrombin therapy have been performed

in animal models for preeclampsia (Shinyama et al 1996a,

Shinyama et al 1996b) . Additional animal studies for other elements

involved in the preeclampsia syndrome have been conducted with AT

intervention . Although these studies are not on preeclampsia per se,

they may provide insight into the ability of antithrombin to ameliorate

these elements of the preeclamptic cascade .

Shinyama et al 1996a, Antithrombin III Prevents Blood

Pressure Elevation and Proteinuria Induced by High

Salt Intake in Pregnant Stroke-Prone Spontaneously

Hypertensive Rats, Biol. Pharm. Bull. 19, 819-823.

In the first model (Shinyama et al 1996a), salt-loading (2% NaCl

diet) in pregnant stroke-prone spontaneously hypertensive

rats caused symptoms similar to those of human preeclampsia

(hypertension and proteinuria) . Antithrombin (60 or 300 U/kg/d)

or saline was administered intravenously for 10 days from days

9-11 to 18-20 (Figure 6) . Blood pressure was measured using an

automated device by the tail-cuff method on days 0, 6-8 and 15-

17 of gestation . Urine was collected for 24 hours pre-conception

and on days 8-10 and 17-19 .

There was a significant elevation of systolic blood pressure (SBP) on

days 15-17 and of urinary protein excretion on days 17-19 of gestation

in salt loaded rats compared to control rats fed a normal diet .

Intravenous administration of antithrombin (60 or 300 U/kg/d) for

10 days from days 9-11 to 18-20, attenuated the aforementioned

changes in blood pressure (Figure 7) and proteinuria (Figure 8) in a

dose dependent manner .

Blood pressure measurement

Urine collection pre-conception

Mating

6-8

8-10 17-19

0 15-17 18-20 (Days of gestation)

Arterial blood sampling

Historical analysisHigh salt intake

Saline or AT III administration

FIGURE 6 Experimental Design (Adapted from Shinyama et al 1996a)


FIGURE 7 Changes in Systolic Blood Pressure (Adapted from Shinyama et al 1996a)

FIGURE 8 Changes in Proteinuria (Adapted from Shinyama et al 1996a)

100

80

60

40

20

0

Day 17-19Day 18-10Pre-conception

U p

rote

in V

(mg/

kg/2

4 h)

*

*

†

Urine was collected for 24h each time.

Saline-treated animals fed normal diet (n=8)

Saline-treated animals fed high-salt diet (n=9)

AT III-(60U/kg/day) treated animals fed high salt-diet (n=8)


Values are + S.E.M.

p<0.05 compared with value for saline-treated animals fed normal diet (Student's t-test)

p<0.05 compared with value for saline-treated animals fed high-salt diet (Dunnett's test)

*

†

Days of Gestation

SBP

(mm

Hg)

260

240

220

200

0 6-8 15-17

**

†

Blood pressure was measured using the tail-cuff method on days 0, 6-8 and 15-17 of gestation.

Saline-treated animals fed normal diet (n=8)

Saline-treated animals fed high-salt diet (n=9)



Salt-loading (except animals fed normal diet)

Saline or AT III administration i.v.

Values are + S.E.M..

p<0.01 compared with value for saline-treated animals fed normal diet (Student's t-test)

p<0.05 compared with value for saline-treated animals fed high-salt diet (Dunnett's test)

**

†

AT also prevented the occurrence of arteriosclerosis and

thickening of the capillary basement membrane of the kidney .

Since the changes induced in these rats by salt-loading were not

due to activation of the blood coagulation system, the effect of AT

in this model resulted from a mechanism largely independent of

its anticoagulant action .

Shinyama et al 1996b Antithrombin III Prevents Renal

Dysfunction and Hypertension Induced by Enhanced

Intravascular Coagulation in pregnant Rats: Pharmacological

Confirmation of the Benefits of Treatment with Antithrombin

III in Preeclampsia, J. Cardiovascular Pharm. 27: 702-711.

In the second animal model (Shinyama et al 1996b), intravascular

coagulation was induced in gestation day 16-20 pregnant rats

by the intravenous administration of tissue thromboplastin (TP)

through the left ventricle of the heart . One hour later, organ blood

flow was measured by the radioactive (37Co-labeled) microsphere

method and fibrin deposition in organs was measured by

radiolabeling with [125I]fibrinogen injected before the TP infusion .

TP, a lipoprotein that initiates the extrinsic coagulation pathway,

caused fibrin deposition in the kidney, lung and liver, but not in

the myometrium and placenta, and produced an 80% decrease

in renal blood flow (RBF), with oliguria and proteinuria . TP also

caused an increase in blood pressure (BP) accompanied by an

increase in plasma renin activity (PRA) .


FIGURE 9 Experimental Design (Adapted from Shinyama et al 1996b)

I-Fibrinogen, i.v.

Surgery

Co-microsphere, i.l.v.

Urine collection (1st period)

Saline or drug injection followed by continuous infusion i.v.

Tranexamic adic200 mg/kg. i.v.

(2nd period)

Thrombopisstin saline9 mL/kg/hr, i.l.v.

0h 1h 2h

Blood pressure measurement

perfusion of heparinized saline from the left ventricle of the heart

intra-left ventricular

intravenously

Once it was established that TP increased fibrin deposition,

proteinuria and blood pressure in the rats, the experiment was

repeated using a prophylactic administration of AT or saline . Five

minutes prior to the TP infusion in some animals, a bolus dose of AT

(60 or 300 U/kg) or saline was administered intravenously followed

by an AT infusion of 30 or 150 U/kg/2h, or saline, respectively .

The administration of AT in this rat model prevented the changes

in fibrin deposition (Figure 10), urinary protein content (Figure 11)

and blood pressure (Figure 12) in a dose-dependent manner . AT

infusion also increased placental blood flow .


FIGURE 10 Fibrin Deposition in Kidney, Liver and Lung (Adapted from Shinyama et al 1996b)

Inhibition by antithrombin III (AT III) of thromboplastin (TP)-induced enhanced fibrin deposition (top) and decreased blood flow (bottom) in the kidney, liver and lung. Fibrin deposition and blood flow in organs were measured 1h after discontinuation of TP infusion. Fibrin deposition in organs was measured with [125l] fibrinogen. Blood flow was measured by the radioactive (57Co) microsphere method. Saline intravenously (i.v.) + intra-left ventricular (i.l.v.) infusion of saline (n=7, white columns), saline i.v. + i.l.v. infusion of TP (n=7, solid orange columns).

1.5

1.0

0.5

0

125 l-F

ibrin

dep

ositi

on(o

rgan

/blo

od ra

tio)

Kidney Liver Lung

**

†

‡

0.15

0.05

0

125 l-F

ibrin

dep

ositi

on(o

rgan

/blo

od ra

tio)

**

†

*

1.5

1.0

0.5

0

Bloo

d flo

w(m

l/g/m

in)

Bloo

d flo

w(m

l/g/m

in)

*

††

0.4

0.2

0

Values are mean + SEM.

p<0.05 and ** p<0.01 as compared with the value for saline-treated animals not receiving TP infusion (Dunnett’s test).

p<0.05 as compared with the value for saline-treated animals receiving TP infusion (Dunnett’s test).

Thromboplastin

*

†


The authors suggest that intravascular coagulation plays a

significant role in the events of preeclampsia and that AT may

have therapeutic potential in its treatment . Also since AT improved

placental blood flow, it may help ameliorate the ischemia that is

occurring in the placenta .

Effect of AT on Prostacyclin Production, TNF-alpha

and Apoptosis

A series of in vitro and in vivo studies have been performed with

antithrombin that demonstrates its ability to increase prostacyclin

(PGI2) production, inhibit TNFα production and apoptosis in

model systems . Prostacyclin is an important mediator of the anti-

inflammatory effect of AT and is a potent inhibitor of leukocyte

activation and monocyte production of TNF-alpha and the

production of IL-8, two pro-inflammatory cytokines .

Mizutani et al (2003) used a rat renal ischemia/reperfusion (I/R) model

to show that antithrombin inhibited leukocyte activation through

promotion of an increase in prostacyclin (an inhibitor of leukocyte

activation) production . A 1998 report from Okajima showed a similar

effect of AT on inhibition of leukocyte activation through induction

of production of prostacyclin in a rat endotoxin-induced pulmonary

vascular injury model .

Harada et al (2004) used a rat hepatic ischemia/reperfusion (I/R)

system to show that antithrombin induced an increase in prostacyclin

production . AT treatment also increased hepatic tissue blood flow

and inhibited hepatic inflammatory responses . The effects of AT

were reversed by pretreatment with indomethacin a non-selective

inhibitor of cyclooxygenase . The authors suggested that AT might

be acting in this system by increasing the production of prostacyclin

and PGE2 through activation of COX-1 . Additionally, AT inhibited TNFα

production . The authors concluded that these effects induced by AT

may contribute to it anti-inflammatory activity .

Hirose et al (2004) demonstrated in a rat model of ischemia/

reperfusion-induced spinal cord injury that AT reduces the injury by

attenuating the inflammatory response . Microinfarctions of the spinal

cord were markedly reduce by AT . AT treatment also increased the

spinal cord tissue levels of 6-keto-PFGIα, a stable metabolite of

FIGURE 11 Urinary Concentration of Protein (Adapted from Shinyama et al 1996b)

FIGURE 12 Changes in Blood Pressure (Adapted from Shinyama et al 1996b)

30

20

10

0

Urin

ary

prot

ein

conc

entr

atio

n(m

g/m

l)

†

Thromboplastin

(TP) was infused during first 1 h period.

Saline intravenously (i.v) + inta-left ventricluar (i.l.v) infusion of saline (n=7)

Saline i.v. - i.l.v. infusion of TP (n=6)

Antithromin III (AT III) (60 U/kg = 30 U/kg/2 h i.v) + i.l.v. infusion of TP (n=7)

AT III (300 U/kg + 150 U/kg/2 h i.v. ) + i.l.v. infusion of saline (n=6)

Values are mean + SEM.

p < 0.01 as compared with the value of saline-treated animals not receiving TP infusion (Dunnett’s test)

p < 0.05 as compared with the value for saline-treated animals receiving TP infusion (Dunnett’s test.)

**

†

Mea

n Bl

ood

Pres

sure

(mm

Hg)

125

100

175

150

75

Time (h)0 1 2

Changes in mean blood pressure of animals during and after thromboplastin (TP) infusion. Blood pressure was measured by pressure transducer through a catheter inserted in the femoral artery.

Saline intravenously (i.v) + intra-left ventrucular )i.l.v) infusion of saline (n=7)

Saline i.v. + i.l.v. infusion fo TP (n=6)

Antithrombin III (AT III, 60 U/kg + 30 U/kg/2 h i.v) + i.l.v. infusion of TP (n=7)

AT III (300 U/kg +150 U/kg/2 h i.v.) + i.l.v. infusion of TP (n=7)

AT III (300 U/kg +150 U/kg/2 h i.v.) + i.l.v. infusion of Saline (n=6)

Values are mean + SEM. *p < 0.05 as compard with the value for saline-treated animals not receiving TP infusion (Dunnett’s test).


prostacyclin . I/R-induced increases in the levels of TNFα, IL-8 and

myeloperoxidase were inhibited by AT treatment . In contrast, Trp49

modified AT (which is not capable of promoting the endothelial

release of prostacyclin since it lacks affinity for heparin) did not

show any protective effects and pretreatment with indomethacin

significantly reversed the protective effect of AT . Collectively,

these observations suggested that AT might reduce I/R-induced

spinal cord injury mainly by its anti-inflammatory effect through

promotion of endothelial production of prostacyclin .

Uchiba et al (2004) showed in the cultured human umbilical vein

endothelial cell (HUVEC) model that AT inhibited TNFα-induced

endothelial cell activation .

Harada et al (2008) showed in an ischemia/reperfusion-induced liver

injury model in mice that AT treatment may prevent reperfusion-

induced hepatic apoptosis by enhancing IGF-1 production .

Pretreatment with anti IGF-1 antibody completely reversed the

therapeutic effects of AT in the wild-type mice .

In Vitro Studies Related to AT as an Anti-inflammatory Agent

AT levels are substantially decreased in patients with severe sepsis or

DIC (such as seen in preeclampsia) by virtue of its consumption during

complex formation with clotting factors and by degradation by elastase .

Additionally, dysfunction of endothelium results in an enhanced

leukocyte rolling and diapedesis into the vascular intima, leading to

edema formation and tissue injury . AT affects this leukocyte function

in an anti-inflammatory way via heparan sulfate proteoglycans .

Conclusions Regarding Non-clinical Studies Supporting

the Use of AT for Treatment of Preeclampsia

Collectively, there is a significant body of research on the

anticoagulant and potent anti-inflammatory properties of

antithrombin (hpAT) . In addition, in animal models for

preeclampsia, AT treatment ameliorated the hypertension,

proteinuria and fibrin deposition which are the key hallmarks of

the preeclamptic syndrome . Since preeclampsia does not occur

spontaneously in quadruped animals and no animal models exist

that fully mimic all features of the multifactorial preeclamptic

syndrome, the next step is testing of AT in human clinical trials in

pregnant women with preeclampsia .

Antithrombin Replacement in Human PreeclampsiaThere have been limited case reports and studies using

antithrombin replacement in the setting of preeclampsia . Buller

administered a dose of 2,000 Units of antithrombin (plasma)

concentrate to an antithrombin deficient patient with severe

preeclampsia and found that the antithrombin infusion improved

blood pressure, proteinuria and coagulation parameters . The

patient had an uneventful Cesarean delivery (Buller 1980) .

Terao et al (1989) reported on 40 patients with preeclampsia . Twenty

seven were treated with antithrombin (plasma) replacement and

13 were untreated . It is unclear how many patients experienced

severe preeclampsia . Antithrombin was dosed at 1,000-2,000 units/

day . In Japan, hypertensive patients are scored on a Gestosis Index

(GI), consisting of edema, proteinuria, systolic and diastolic blood

pressure criteria . The authors considered that there was a 40%

efficacy in the treated group vs 0% in the untreated group . They

also found a correlation between GI and antithrombin activity .

Nakabayashi and colleagues evaluated antithrombin (plasma)

replacement vs heparin in early onset severe preeclampsia

with intrauterine fetal growth restriction under 32 weeks

(Nakabayashi et al 1999) . Fifteen patients received a dose of

antithrombin replacement 1500IU/day x7 days . The antithrombin

infusion was associated with improved systolic blood pressure and

sonographic estimation of fetal weight, as compared to heparin .

The authors concluded that antithrombin replacement therapy

was useful for improving maternal hypertension and fetal weight

in severe preeclampsia .

Kobayashi and colleagues performed a Phase II trial with 29

patients with severe preeclampsia (Gestosis index≥ 6), at 24-36wks

(Kobayashi et al 2003a) . The study was designed to assess the

efficacy of antithrombin plasma concentrate, as a coagulation

inhibitor for the treatment of severe preeclampsia . Antithrombin

replacement at 1,500 units plus heparin 5000 units daily was

compared to a control group who received only heparin 5000

units daily for 7 days . The Gestosis Index and biophysical profile,

a measurement of fetal well being, were both significantly

improved with antithrombin replacement (p=0 .46 and p=0 .022,

respectively) . When the authors compared coagulation parameters,

antithrombin replacement was associated with improved levels of

plasmin- plasmin inhibitor complex, D-Dimer, and platelet counts .


Protocol of monitoring and line of treatment

First ApproachAT III concentrates to the purpose to maintain range over 120%.

General Approach

Fresh Frozen Plasma (from 10 to 20 ml/kg), AT III at high doses (3000-4000 IU.day), FIBRINOGEN only if under 1.25 g/lt

Platelets if number under 70,000, No PPSB

FIGURE 13 Protocol for AT Use in Preeclampsia (Adapted from Mangione & Giarratano 2002)

The authors’ impression was that the addition of antithrombin

replacement was superior to heparin alone in improving maternal

and fetal outcome .

Kobayashi reported on a late Phase II study which demonstrated a

linear dose-response relationship for the use of antithrombin (plasma)

concentrate in the treatment of severe preeclampsia . He reported

improved uteroplacental circulation found with administration

of antithrombin replacement . The optimal dose of antithrombin

replacement was 3000 units/ day, which was efficacious and safe for

both mother and fetus (Kobayashi 2003b, 2005) .

Maki and colleagues performed a controlled double blind phase III

trial, with 133 patients with severe preeclampsia, whose gestational

ages ranged from 24-35wks (Maki et al 2000) . The Gestosis Index

was ≥ 6 . Sixty six patients received antithrombin replacement at a

dose of 3000 IU per day . Sixty seven patients received intravenous

placebo for seven days . The patients were followed for 2 weeks until

delivery . The authors found a significant prolongation of pregnancy

with antithrombin replacement (16 .8 +/-2 .0 v 10 .2 +/- 1 .2, p .007,

and a significantly greater gestational age at delivery 34 .1 +/- 3 .2 vs

33 .0 +/- 2 .7wks . The study demonstrated a significant mean increase

in gestational age of 6 .5 days in the antithrombin replacement group .

Both antithrombin antigen and antithrombin activity increased

significantly (p value .001) in the antithrombin replacement group,

compared to placebo . The authors concluded that antithrombin

replacement in addition to conventional therapy improved maternal

symptoms, improved the biophysical profile score, prolonged

pregnancy and decreased prevalence of very low birth weight infants .

Paternoster performed a dosing study comparing 2 different

dosing regimens of antithrombin (plasma) replacement, in the

setting of severe preeclampsia between 24-33 wks (Paternoster et

al 2004) . The High dose antithrombin group (n=10) received 3000

IU/d x5 day (1268 .000 IU total; 12600IU per pt mean dose) . The

Standard dose antithrombin group (n=13) received antithrombin

replacement to maintain 80% antithrombin activity . They found

that the Standard dose group received 43 .800 IU total dose per

patient, and a mean dose per patient of 3370 IU . The High dose

group ultimately received 3 .7x higher dose than the Standard

dose group . The mean number of days the pregnancy was

prolonged in the High dose group was 6 days, as compared to 3 .5

days in the Standard dose group . The High dose group also had

greater birthweights, (1185g vs 1005g), but this difference was not

significant (p 0 .3) . Their conclusion was that the Standard dose of

antithrombin replacement corrects the hemostatic abnormality,

while the High dose also corrects inflammatory state .

In summary, there is preliminary evidence suggesting a benefit

to antithrombin replacement in the setting of preeclampsia .

The published studies do suggest a benefit to antithrombin

replacement in preeclampsia . There is a disproportionate

contribution to the literature by the Japanese studies which

are confounded by a preeclamptic classification not used by

the rest of the developed world . The preliminary data does not

suggest harm, either for the mother or the fetus . A high quality

trial is needed to address the many questions raised by these

provocative preliminary studies .

Four additional clinical publications that advocate the use of AT in

clinical studies in preeclampsia, are available . The first study is a PK

study conducted by Weiner et al (1990) in 5 normal healthy and 5

preeclamptic pregnant women with a documented deficiency of

AT . Healthy pregnant women received 1500 units of antithrombin

(plasma) intravenously over 20 min . Due to their low baseline AT

activity of 61 to 75% of normal, preeclamptic pregnant women

received 3000 units of AT intravenously . Serial blood specimens

were obtained over the next 12 hr from both groups of women .

The mean t1/2

of AT in the healthy group was 29 .4h ±3 .4h, whereas

the mean t1/2

in the preeclamptic group was 8 .5h ±1 .2h . The

significantly faster clearance in the preeclamptic group suggests

rapid consumption of AT in this disorder .

The second publication by Mangione and Giarratano (2002)

reports on the measurement of inflammatory cytokines (IL-6, TNF,

IL-10), antithrombin, Protein C and Tissue Factor Pathway Inhibitor

(TFPI) in the plasma of 36 patients with normal pregnancy and

severe preeclampsia . They also measured an Organ Dysfunction

Score (0-15) . A correlation was found particularly between AT

levels and the clinical scores . The authors indicate that they have

in place in their ICU a monitoring and treatment protocol for

preeclamptic patients that specifies replacement of the AT levels

in the preeclamptic patient to at least 120% of normal (Figure 13) .

They also state that a multi-center study on AT supplementation

in preeclampsia was ongoing, although to date no follow-up

publication has been forthcoming with the study results .


A third publication by Marietta et al (2009) reported on the

measurement of antithrombin, platelet, fibrinogen and D-dimer

levels in women with preeclampsia for several days over the time of

delivery . They report that a significant drop in AT levels is associated

with the clinical worsening of preeclampsia leading to the need for

delivery, regardless of its severity . The AT drop was associated with

a drop in platelet count and fibrinogen levels, whereas D-dimer

fluctuations were not statistically significant . The AT levels in these

individuals recovered immediately after parturition . AT was the only

coagulation factor measured that displayed this temporal recovery

after parturition . They conclude that these results strengthen the

support for AT interventional trials in preeclampsia .

The last publication is a review article by Haram et al (2009) on

the HELLP syndrome, which is a variant of preeclampsia . This

complication of pregnancy is characterized by hemolysis, elevated

liver enzyme and low platelet count (HELLP) and occurs in 0 .5

to 0 .9% of all pregnancies and in 10–20% of cases with severe

preeclampsia . The authors suggest that antithrombin (plasma)

is a possible therapeutic option for treatment of preeclampsia

due to it demonstrated ability to correct hypercoagulability,

stimulate prostacyclin production, regulate thrombin-induced

vasoconstriction and improve fetal status and fetal growth . They

suggest that antithrombin should be tested in randomized studies

in women with the HELLP syndrome .

Rationale for Use of Antithrombin in PreeclampsiaFrom the medical research described in the literature, it is clear

that preeclampsia is a multifactorial disorder that involves both the

cardiovascular and inflammatory systems . Due to its pleiotropic

nature as an anticoagulant and anti-inflammatory agent, AT has

been suggested as a potential treatment to ameliorate this disorder .

If one considers the various features of the preeclamptic state

(described and summarized in this document) and the potential

impact that AT treatment might provide on these features (Table 1),

it is clear that the anti-inflammatory properties of AT would most

likely provide the most benefit in this disorder . However, one cannot

underestimate the damage that thrombin and microthrombi play

in placental damage and maternal cardiovascular damage .

TABLE 1 Rationale for Use of AT in Preeclampsia

Observed in Preeclampsia (PE) Potential Impact of AT Treatment

Reduced levels of antithrombin thereby reducing anticoagulation; correlates with worsening of preeclampsia

Increase antithrombin levels to normal or supranormal levels

Elevated TAT levels Should provide additional elevation in TAT levels due to hyper-coagulant state in PE

Placental infarctions due to increased thrombosis Inhibits/reduces thrombosis

Fibrin deposition in maternal organs and placenta Inhibits fibrin deposition

Increased thromboxane A2 production and decreased

prostacyclin production, which provides a thromboxane A

2/prostacyclin ratio which favors the promotion of

vasospasm, induces supplementary platelet aggregation and endothelial damage

Increased production of prostacyclin by AT binding to heparan sulfate proteoglycans on the surface endothelial cells, which would rebalance the thromboxane A

2/prostacyclin

ratio and ameliorate vasospasm, platelet aggregation and endothelial damage

Increased thrombin-induced vasoconstriction Increased blood pressure

Regulates thrombin-induced vasoconstriction Normalize blood pressure

Increased proteinuria Reduces proteinuria

Increased neutrophil adhesion to the endothelium Reduces neutrophil adhesion to endothelium

Release of pro-inflammatory cytokines such as TNFα and IL-6 and IL-8 that mediated vasoconstriction and vascular damage

Inhibits production of the pro-inflammatory cytokines

Increased IL-6 release, which increases endothelial permeability Significantly inhibits production of IL-6 in inflammatory disorders

Significant platelet dysfunction Reduces platelet dysfunction

Decreased placental blood flow leading to ischemia Increased placental blood flow


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A significant body of research, both non-clinical and clinical,

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AT therapeutic treatment of women with preeclampsia . As

suggested in a number of the clinical studies cited above,

the next step is the use of antithrombin in a well-controlled,

randomized clinical study in women with preeclampsia .


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Antithrombin and Preeclampsia