ADVANCES IN CLINICAL CHEMISTRY, VOL. 53

BIOCHEMISTRY OF HELLP SYNDROME

Chiara Benedetto,1 Luca Marozio, Annalisa Tancredi,Elisa Picardo, Paola Nardolillo, Anna Maria Tavella,

and Loredana Salton

Department of Obstetrics and Gynaecology, University ofTorino, Torino, Italy

1. A

1C

006DO

bstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

orresponding author: Chiara Benedetto, e-mail: chbened@tin.it

85

5-2423/11 $35.00 Copyright 201I: 10.1016/S0065-2423(11)53004-5 All

1, Elsevierrights rese

85

2. I

ntroduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

2.1.

C linical Features of HELLP Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

2.2.

L aboratory Findings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

2.3.

M aternal and Perinatal Outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

3. P

athogenesis of HELLP and Preeclampsia: The Role of Placenta . . . . . . . . . . . . . . . . 89

4. I

nflammatory Response in HELLP Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

5. H

ELLP Syndrome, Complement Pathway, and the Coagulation System. . . . . . . . . 95

6. C

onclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

R

eferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

1. Abstract

The HELLP syndrome is a serious complication of pregnancy character-

ized by hemolysis (H), elevated liver (EL) enzymes, and low platelet (LP)

count that occurs in 0.2–0.6% of all pregnancies and in 10–20% of cases with

severe preeclampsia and frequently leads to adverse maternal and perinatal

outcome. The exact pathobiology of HELLP syndrome has not been clearly

defined. As it is considered a form or a complication of severe preeclampsia,

it likely has its origin in aberrant placental development and function result-

ing in ischemia-producing oxidative stress. However, there is still a debate on

whether HELLP must be considered a severe form of preeclampsia or a

separate disease entity. It can be described as a placenta-induced disease, as

is preeclampsia itself, but with a more acute and predominant inflammatory

Inc.rved.

86 BENEDETTO ET AL.

process typically targeting the liver and with a greater activation of the

coagulation system. This occurs during a disordered immunologic process

and may be due to a genetic predisposition. In this review, we discuss the

main biochemical characteristics of HELLP syndrome, particularly focusing

on molecular aspects of placental involvement and maternal systemic

responses.

2. Introduction

The HELLP syndrome is a serious complication of pregnancy character-

ized by hemolysis (H), elevated liver (EL) enzymes, and low platelet (LP)

count occurring in 0.2–0.6% of all pregnancies and in 10–20% of cases with

severe preeclampsia [1]. In about 70% of the cases, the HELLP syndrome

develops before delivery with a peak frequency between the 27th and 37th

gestational weeks; 10% occur before the 27th week, and 20% beyond the 37th

gestational week [2,3]. In some cases, it develops in the postpartum period,

usually within the first 48h in women who have had proteinuria and

hypertension prior to delivery [2].

2.1. CLINICAL FEATURES OF HELLP SYNDROME

The majority of women with the HELLP syndrome have had hypertension

and proteinuria, which may be absent in 10–20% of the cases [4]. The more

common symptom at presentation is right upper abdominal quadrant or

epigastric pain, nausea, and vomiting being less frequent [4]. Up to 30–60%

of women have headache; about 20% have visual disturbances and other

features of severe preeclampsia or impending eclampsia [4]. The clinical

symptoms often precede the laboratory findings. In some cases, however,

the HELLP syndrome may present with nonspecific viral syndrome-like

symptoms or malaise [4].

The syndrome, which is considered a complication of preeclampsia, is

characterized by prominent endothelial cell damage within the liver.

Hypovolemia is suggested with a decrease in the liver blood flow on Doppler

examination in patients with preeclampsia, who have subsequently devel-

oped HELLP syndrome [5]. Hepatic ischemia may cause infarction, subcap-

sular haematomas, and intraparenchymatous hemorrhage, resulting in

hepatic rupture, a rare but severe and life-threatening complication [6].

Recurrent episodes of hepatic haematoma and rupture in subsequent preg-

nancies have been reported, suggesting that there may be a specific predispo-

sition to this condition [7]. On liver biopsy, periportal hemorrhage, focal

parenchymatous necrosis, and macrovesicular steatosis may be observed in

BIOCHEMISTRY OF HELLP SYNDROME 87

up to one-third of patients. Fibrin and hyaline deposits are seen by immuno-

fluorescence at the level of liver sinusoids. However, there is little correlation

between histological findings and clinical presentation [8].

Hemolysis, one of the major characteristics of the disorder seen on a

microangiopathic blood smear, reflects the damage of the vascular endothe-

lium. Red cell fragmentation represents the extent of small vessel involve-

ment, and schizocytes and Burr cells are usually present [9]. Polychromatic

red cells are also seen in blood smears, and increased reticulocyte counts

reflect the compensatory release of immature red cells into peripheral blood.

Decreased PLT count in the HELLP syndrome is due to their increased

consumption. Platelets are activated and adhere to damaged vascular endo-

thelial cells, resulting in increased platelet turnover with shorter lifespan [9].

2.2. LABORATORY FINDINGS

Hemolysis causes increased serum lactate dehydrogenase (LDH) levels and

decreased hemoglobin concentrations [10]. Free hemoglobin is converted to

unconjugated bilirubin in the spleen or may be bound by haptoglobin. The

hemoglobin–haptoglobin complex is cleared quickly by the liver, leading to

decreased haptoglobin plasma levels [10,11]. Low haptoglobin concentration

is the preferred marker of hemolysis [12]. Thus, the diagnosis of hemolysis is

supported by high LDH concentration and the presence of unconjugated

bilirubin, but the demonstration of low or undetectable haptoglobin concen-

tration is a more specific indicator. Intravascular hemolysis is diagnosed by

abnormal peripheral blood smear, increased serum bilirubin (�20.5 �mol/L

or �1.2 mg/100 mL), and elevated LDH levels (>600 units/L (U/L)) [13,14].

However, according to Smulian et al., the threshold of normal LDH values

may be much lower than 600 U/L depending on the laboratory method

adopted [15].

Elevation of liver enzymes may reflect the haemolytic process as well as

liver involvement. Enhanced aspartate aminotransferase (AST) and alanine

aminotransferase (ALT) levels are mostly due to liver injury [10]. Visser and

Wallenburg used ALT >30 U/L to define abnormality (2 SD above mean)

[16], while Sibai suggests a cutoff value for ALT >70 U/L [4].

Thrombocytopenia (platelets < 100,000/ml) is obligatory in the HELLP

syndrome. Reduced platelet count in pregnancy may be also caused by

gestational thrombocytopenia, immune thrombocytopenic purpura, and

preeclampsia [17], but in those cases, hemolysis and liver damage are usually

absent.

Sometimes, the differential diagnosis of HELLP from ‘‘acute fatty liver of

pregnancy’’ (AFLP) may be very difficult. AFLP is a rare but life-threatening

complication mainly of the third trimester. The patient usually presents with

88 BENEDETTO ET AL.

a 1- to 2-week history of malaise, nausea, vomiting, epigastric or right upper

quadrant pain, headache, or jaundice, more rarely with hepatic encephalop-

athy or coma. Signs of preterm labor and/or fetal demise may be present.

Other findings may include hypertension, proteinuria, low-grade fever,

ascites, and bleeding from severe coagulopathy [18]. Common laboratory

findings in AFLP are hemoconcentration, elevated white blood count, and

normal or reduced platelet count. The production of antithrombin, fibrino-

gen, and coagulation factors by the liver is significantly reduced, often

leading to disseminated intravascular coagulation. Serum electrolytes will

reveal evidence of metabolic acidosis with elevated creatinine and uric acid

values [19]. Blood sugar is usually low, but may be high in association with

pancreatitis. Liver enzymes (AST, ALT, alkaline phosphatase) and bilirubin

will be elevated. The increase in bilirubin is mainly of the conjugated form,

with levels usually higher than 5 mg/dL. Ammonia levels increase in the late

stage of the disease [20]. Ultrasonography, CT, and MRI are not sufficiently

sensitive to confirm or exclude the diagnosis, and the gold standard for

confirming the diagnosis of AFLP is the liver biopsy with special stains for

fat, such as red oil: histopathological findings reveal swollen, pale hepato-

cytes with central nuclei. However, liver biopsy is rarely used in the clinical

practice [18]. Prompt delivery, within 24 h from the diagnosis and after

maternal stabilization, is a reasonable approach. In general, most patients

with AFLP will start to improve 2–3 days after delivery. However, in some

cases, deterioration in liver and renal function, encephalopathy, and coagu-

lopathy may continue, requiring life-supporting treatments. In rare cases,

liver transplantation will be required [21].

2.3. MATERNAL AND PERINATAL OUTCOME

The HELLP syndrome is associated with both maternal and neonatal

complications. Spontaneous rupture of a subcapsular liver haematoma in

pregnancy is a rare but life-threatening complication that occurs in less than

1% of the cases with the HELLP syndrome. Rupture most often occurs in the

right liver lobe [4, 22–24]. More common and serious maternal complications

are abruptio placentae, disseminated intravascular coagulation, and

subsequent severe postpartum bleeding, retinopathy, cerebral bleeding, and

stroke [25–32]. In a large retrospective cohort study comprising 442 preg-

nancies complicated by the HELLP syndrome, the maternal mortality was

1.1% [2], which is in accordance with other reports [4,33,34]. Isler et al. found

cerebral hemorrhage or stroke to be the primary cause of death in 26% and

the most contributing factor in another 45% of the deaths [35]. Maternal

mortality rate in hepatic rupture ranges from 18% to 86% [8].

BIOCHEMISTRY OF HELLP SYNDROME 89

Perinatal mortality and morbidity are considerably high in the HELLP

syndrome and are primarily dependent on the gestational age at onset

[36,37]. The perinatal mortality rate related to the HELLP syndrome is

between 7.4% and 34% [4,38,39]. Neonates delivered before completion of

32 weeks’ gestation have the highest risk of perinatal death [36,37].

3. Pathogenesis of HELLP and Preeclampsia: The Roleof Placenta

The exact pathobiology of HELLP syndrome has not been clearly defined.

As it is considered a form or a complication of severe preeclampsia, it likely

has its origin in aberrant placental development and function resulting in

ischemia-producing oxidative stress. An abnormal interaction between ma-

ternal and placental tissue at the time of trophoblast implantation is thought

to play a key role in the pathogenesis of preeclampsia. In preeclamptic

pregnancies, trophoblast invasion of the spiral arteries at the time of placen-

tation is confined to the inner layer of the myometrium so that a low

resistance, high flow uteroplacental circulation typical of normal pregnancy

cannot be established, and placental ischemia will develop [40,41]. The

insufficient trophoblast invasion is believed to be the consequence of the

abnormal interaction between decidual immune cells and paternally derived

antigens on trophoblasts’ surface [42]. Owing to generalized vasoconstric-

tion, microthrombi formation in small vessels, and plasma volume reduction,

blood flow is impaired in every organ and tissue. The maternal vascular

endothelium is an early target of placental ischemia, and its activation is

responsible for the generalization of damage [43,44]. The clinical manifesta-

tions of preeclampsia begin with the loss of vascular refractoriness to vaso-

constrictors typical of normal pregnancy, with a subsequent increase in

peripheral resistances. The mechanisms underlying the impaired vascular

reactivity in preeclampsia are not fully understood, but they might be attrib-

uted to the systemic endothelial dysfunction [43,45,46].

The link between abnormal trophoblast invasion, placental ischemia, and

maternal vascular endothelium activation is still unclear. Oxidative stress

following immune rejection of the trophoblast in the decidua seems to play a

key role. The features of endothelial damage in PE are similar to those

observed in other diseases in which the impact of oxidative stress is known,

such as atherosclerosis, diabetes, septic shock, and ischemia–reperfusion

syndrome. In PE, oxidative agents are released into the intervillous spaces

by activated leukocytes and the ischemic placenta itself. Several markers of

oxidative stress significantly increase during PE, even before the clinical onset

of the disease, and endogenous antioxidants sharply decrease [47–49].

90 BENEDETTO ET AL.

Moreover, the placental release and the activity of angiogenic and endothelium-

protecting agents, such as the ‘‘vascular endothelial growth factor’’ and the

‘‘placental growth factor’’ are deeply impaired [50–53].

HELLP syndrome has many features in common with preeclampsia and

can be described as a placenta-induced disease, as is preeclampsia itself, but

with a more acute and predominant inflammatory process typically targeting

the liver and with a greater activation of the coagulation system. This occurs

during a disordered immunologic process and may be due to a genetic

predisposition [8,54,55]. There is still a debate on whether HELLP must be

considered a severe form of preeclampsia or a separate disease entity.

It is well documented that the placenta is a prerequisite for the develop-

ment of both preeclampsia and HELLP syndrome, and it has been thought

that differences in placental gene expression may account for clinical and

molecular differences between the two syndromes. In a recent study, Buimer

et al. investigated differences in gene expression between placental tissue

obtained from normotensive pregnant women and women with preeclampsia

or HELLP syndrome [56]. In their study, first, comparison of serial analysis

of gene expression profiles of 28 weeks’ control placenta (from idiopathic

preterm delivery) to a HELLP/preeclampsia matched for gestational age

identified 404 differentially expressed transcripts. Second, using semiquanti-

tative real-time PCR, the expression levels of 37 of these transcripts

were analyzed in placentas from normal pregnant women and from patients

with HELLP or preeclampsia. Third, nearest centroid classification method

determined the HELLP-specific molecular signature consisting of the

upregulated expression of genes encoding the vascular endothelial growth

factor receptor (Flt1), leptin, pappalysin2, and WW domain containing

transcription regulator 1 (WWTR1), combined with downregulated expres-

sion of the genes encoding cadherin-associated protein (CTNNAL), glutathi-

one S-transferase-p1 (GSTP1), and calgranulin A (S100A8). Four of these

seven genes (Flt1, GSTP1, leptin, and pappalysin2) have been previously

associated with preeclampsia, although not specifically to HELLP syndrome

[57–60]. The altered expression ofWWTR1,CTNNAL1, and S100A8 has not

been associated with placental function or dysfunction previously. The

authors found that this set of seven genes expression discriminates HELLP

placenta from control and preeclamptic placenta with a 24%misclassification

rate (95% CI 8.3–41.9), independent from known risk factors like parity and

ethnicity. Although it is not known the exact role of the placental expression

of these genes in the pathogenesis of preeclampsia and HELLP syndrome,

this finding might suggest that HELLP is not a variant of preeclampsia but a

separate disease entity. It might be suggested that the abnormal placentation

in HELLP syndrome may trigger a specific expression of placental genes

different from those activated in preeclampsia, leading to enhanced local and

BIOCHEMISTRY OF HELLP SYNDROME 91

systemic inflammatory response and endothelium activation, with a particu-

lar involvement of the liver and the coagulation system probably due to

maternal predisposition.

At this regard, it has been recently observed that the allelic and carrier

frequencies of the BclI polymorphism of the glucocorticoid receptor

(GR) gene were significantly higher in women with HELLP syndrome

compared to healthy pregnant women (p ¼ 0.004, OR 2.89) and to those

with severe preeclampsia (p ¼ 0.013, OR 2.56) [61]. Moreover, the BclI

carrier status had a significant impact on clinical laboratory parameters of

women with HELLP syndrome, as the AST, LDH, and ALP levels were

significantly higher, whereas the platelet count tended to be lower in

BclI carriers than in noncarriers. There were no significant differences in

carrier and allelic frequencies of the N363S and ER22/23EK polymorph-

isms of GR gene between groups [61]. Previous studies have demonstrated

that the BclI polymorphism of GR gene results in increased glucocorticoid

sensitivity in vivo and is associated with cardiovascular risk factors and with

autoimmune diseases [62,63]. The finding suggests that BclI polymorphism

of the GR gene may play a role in the pathogenesis of HELLP syndrome

and may account for clinical characteristics of the syndrome, such as EL

enzymes, LP count, and sensitivity to corticosteroids. Since preeclampsia

and HELLP syndrome develop exclusively in human, it seems particularly

interesting that alignment analysis of DNA sequences obtained from

database indicated the absence of the BclI site in six animal vertebral

species [61].

The difference between HELLP and preeclampsia is underlined also by

clinical aspects. For example, women with preeclampsia and no HELLP

proved to have more often a profile consistent with the metabolic syndrome.

Moreover, preeclamptic patients have a fourfold higher prevalence of throm-

bophilia as compared to those who had experienced HELLP [64]. Certain

maternal features known as risk factors for preeclampsia, such as obesity,

have not been associated with the HELLP syndrome [65]. Furthermore,

women with preeclampsia differ from those with HELLP by the presence

of a smaller placenta with more infarcts, and this data support the view of a

longer subclinical disease period preceding preeclampsia as compared to

HELLP [66]. Sep et al. postulate that preeclampsia differs from HELLP by

a more gradual course in early pregnancy due to unfavorable constitutional

conditions for placental growth and development [64]. Eventually, intervil-

lous hypoxia results in the placental release of toxic substances pushing the

subclinical condition into the well-known clinical symptomatology. Con-

versely, an abnormal immune response to the placental allograft with non-

appreciable negative impact on placental function is thought to characterize

the subclinical phase of HELLP. The acute course and appearance of

92 BENEDETTO ET AL.

HELLP, with episodic exacerbations together with the sensitivity to

corticosteroids, suggest a key role for an immune-mediated and prominent

inflammatory response [67].

4. Inflammatory Response in HELLP Syndrome

The maternal signs of HELLP syndrome as hypertension, proteinuria,

intravascular coagulation, LPs, and hemolysis can all be explained by a

systemic inflammatory activity involving a maternal endothelial cell dysfunc-

tion. In many cases, the severity of HELLP syndrome fluctuates, resulting in

a pattern of exacerbations and remissions. During exacerbations, systemic

endothelial activation produces abnormalities due to thrombotic microan-

giopathic hemolysis, with periportal or focal parenchymal necrosis of hepa-

tocytes [13]. This leads to increased plasma levels of AST and glutathione-S-

transferase alpha 1-1 (GSTA1-1), a very sensitive marker for hepatocellular

damage [2,68]. Van Runnard Heimel et al. [55] recently observed that during

a HELLP exacerbation, plasma levels of C reactive protein were significantly

higher than those in normal pregnancy or in preeclampsia and decreased

during remission. In HELLP patients, plasma levels of IL-8 and tumor

necrosis factor-a (TNF-a) were below the detection limit, as in normal

pregnancy and preeclampsia. Plasma levels of IL-1b, IL-10, and sIL-6R

did not differ between groups, neither during exacerbations nor during

remissions of HELLP. Significant differences were found in IL-6 and

IL-1Ra levels. During a HELLP exacerbation, plasma levels of both cyto-

kines were significantly increased as compared with preeclampsia and nor-

mal pregnancy. During HELLP exacerbation, median GSTA1-1 levels were

significantly higher as compared with preeclampsia and normal pregnancy.

The authors concluded that these findings confirm that the development of

HELLP syndrome is associated with a further intensified inflammatory

response. Moreover, they observed that prednisolone therapy in patients

with HELLP syndrome abolished the rise in plasma levels of the cytokine

IL-6 during exacerbation. Since the activated vascular endothelium is an

important source for circulating IL-6, it may be speculated that corticoster-

oids act by stabilizing the endothelium in patients with HELLP as previously

reported [69–71]. These observations are in agreement with the results of

several randomized controlled trials [12] showing a rapid recovery of the

platelet count during corticosteroid therapy in patients with HELLP syn-

drome. This effect may be the result of the decrease of endothelial activation

following corticosteroids administration. In the study of van Runnard Hei-

mel et al. [55], prednisolone had no beneficial effect on the liver damage

commonly seen in HELLP exacerbation, as reflected by an unaltered course

BIOCHEMISTRY OF HELLP SYNDROME 93

of plasma levels of AST, ALT, and GSTA1-1 during prednisolone therapy.

A possible explanation of this finding is that prednisolone does not seem to

impede the formation of microthrombi, the main cause of hepatic damage.

However, a major pathogenic mechanism for liver disease in HELLP syn-

drome is Fas (APO-1, CD95)-mediated apoptosis of hepatocytes [54]. Fas-

ligand was found to be produced in the placenta. Extracts of placenta were

cytotoxic for human hepatocytes and cytotoxic activity increased as HELLP

syndrome developed [72]. It is possible that corticosteroid is not able to

prevent the cytotoxic activity of placental microparticles on the liver.

The role of the placenta in triggering the inflammatory response typical of

HELLP syndrome has been recently investigated by Tranquilli et al. [73].

They analyzed the expression of 96 genes involved in inflammatory response

in the placenta from women with HELLP syndrome and from healthy

women at term and evaluated some cytokines probably involved in impor-

tant steps of the inflammatory response such as transforming growth factor

(TGF)-b, interleukin (IL)-6 Ra, IL-10, IL-16, and CCL-18 and CXCL5

chemokines. Macroarray analysis identified 14 genes encoding differentially

expressed cytokines. Gene expression measurement (HELLP vs. healthy)

revealed a significant upregulation for IL-10, IL-6 receptor, and TGF-b3in HELLP placenta, while the expression of CCL18, CXCL5, and IL-16 was

significantly downregulated. IL-10 has a powerful anti inflammatory effect.

It is important in regulating immune function, by inhibiting macrophages,

reducing antigen-specific T-cells proliferation, and diminishing the antigen-

presenting capacity of monocytes via downregulation of class II major

histocompatibility complex expression [74]. It has also been reported that

IL-10 has an important inhibitory role in regulation of T-cell responses and

acute inflammation, and it downregulates matrix metalloprotease (MMP)-9

[74]. Its overexpression in HELLP placenta may be regarded as a compen-

satory mechanism against excessive inflammatory response. In HELLP

placenta, the expression of IL-6 receptor (IL-6 Ra) is increased. IL-6, a

cytokine normally produced at the maternal–fetal interface, stimulates

MMP-2 and MMP-9. IL-6 Ra produces a signal transduction occurring

through two pathways: the Ras/mitogen-activated protein kinase (MAPK)

pathway and the Jak/Stat pathway, thus making it an important control in

the inflammatory response. It is known that dysregulation of IL-6-type

cytokine signaling contributes to the onset and maintenance of several

inflammatory and autoimmune diseases, such as rheumatoid arthritis, in-

flammatory bowel disease, osteoporosis, and multiple sclerosis [75].

The TGF-b family is involved in cellular proliferation and differentiation,

extracellular matrix modification, tissue remodeling, and angiogenesis.

It has been observed that TGF-b3 is overexpressed in preeclamptic placen-

tas, and its inhibition restores the invasive capability of extravillous

94 BENEDETTO ET AL.

trophoblasts [76]. In HELLP placenta, TGF-b3 was 2.5-fold upregulated as

compared to placenta from normal pregnancy, and this suggests an involve-

ment in pathogenic mechanisms of HELLP syndrome. Chemokines are

implicated in angiogenesis, cell recruitment, and lymphoid trafficking and

can modulate innate and adaptive immune response [77]. The chemokines

decrease observed in HELLP placenta may be involved in the derangement

of immune activity control at the maternal–fetal interface. Finally, since

IL-16 has been linked with modulation of Th2 cell-mediated inflammation

in response to allergens [78], the decrease of IL-16 expression in HELLP

placenta suggests a role of the cytokine in the immune and inflammatory

features of the syndrome.

In preeclampsia, there is exacerbation of physiological changes associated

with pregnancy such as insulin resistance, altered immune responses, and

inflammatory pathway activation. These exaggerated responses seen in pre-

eclampsia are reminiscent of metabolic syndrome and also are evident in

gestational diabetes mellitus. Many features of the insulin resistance syn-

drome have been associated with this condition. These include hypertension,

hyperinsulinemia, glucose intolerance, obesity, and lipid abnormalities.

Other accompanying abnormalities may include elevated levels of leptin,

TNF-a, tissue plasminogen activator, plasminogen activator inhibitor-1

(PAI-1), and testosterone. The documentation of these features before the

onset of preeclampsia suggests that insulin resistance or associated abnorm-

alities may have a role in this disorder. Furthermore, the recognition that

features of the insulin resistance syndrome persist many years after

pregnancy among women with preeclampsia raises the possibility that these

women may have increased risk for future cardiovascular disease. These

observations suggest that interventions to reduce insulin resistance may

reduce the risk of both hypertension in pregnancy and later life cardiovascu-

lar complications [79]. The link between insulin resistance and preeclampsia

is not clear but novel findings providing some insight have been reported

recently. Inositol phosphoglycan P-type (P-IPG) in preeclampsia has been

extensively investigated and increased production has been demonstrated.

This molecule acts as a second messenger of insulin, enhances the metabolic

effects of insulin, and is associated with insulin resistance. An increase

in urinary release of P-IPG during pregnancy may herald the onset of

preeclampsia. The overexpression of P-IPG during preeclampsia may be a

counter-regulatory mechanism to insulin resistance since these molecules

mimic insulin action. Further knowledge about the nature of the metabolic

syndrome during preeclampsia and the degree of association between its

components will help to inform future research efforts. To date, there are

no studies specifically aimed to investigate the link between insulin resistance

and HELLP syndrome [80].

BIOCHEMISTRY OF HELLP SYNDROME 95

5. HELLP Syndrome, Complement Pathway, andthe Coagulation System

Evidence from the literature supports a role for the complement system in

the pathogenesis of preeclampsia/HELLP. Increased levels of the comple-

ment alternative pathway (CAP) activation fragment Bb early in pregnancy

were associated with the subsequent development of preeclampsia [81].

Complement activation was shown to induce dysregulation of angiogenic

factors and to cause fetal rejection and growth restriction in a murine model

of spontaneous miscarriage and intrauterine growth restriction [82]. Com-

plement activation products, particularly C5a, stimulate monocytes to pro-

duce sVEGFR-1 (sFlt-1) and thereby sequester VEGF [80], and it is known

that increased release of sFlt-1 from ischaemic placenta is one of the key

pathogenic mechanisms in the development of preeclampsia/HELLP syn-

drome and in the endothelial activation typical of the diseases [50]. More-

over, it has been observed that some SNPs polymorphisms of VEGF,

particularly the VEGF �460TT and the þ405CC genotypes, may increase

the risk for HELLP syndrome [83].

HELLP syndrome shares several clinical and biological features with

thrombotic microangiopathy (TMA). TMA is characterized by the occur-

rence of thrombi in the microvasculature of several organs, leading to

thrombocytopenia, mechanical haemolytic anemia, and end-organs failure

[84]. TMA may be classified into three main subtypes: ADAMTS-13

deficiency-related TMA, complement dysregulation-related TMA, and inde-

terminate TMA [85]. Previous studies have shown that HELLP is not asso-

ciated with a deficiency in ADAMTS-13 activity [86], which is a disintegrin

and metalloprotease that cleaves the high molecular weight von Willebrand

factor oligomers secreted by endothelial cells, resulting in unusually large

multimers that can aggregate to form microvascular platelet thrombi [87].

In a recent study, Fakhouri et al. [88] evaluated the plasma concentration of

factor H (FH), factor I, C3, C4, and factor B in 11 patients with HELLP

syndrome. They also analyzed the expression of membrane cofactor protein

(MCP) and the coding sequences for complement factor H (CFH) and

complement factor I (CFI). They identified four patients with a mutation

in one of the genes coding for proteins involved in the regulation of the

alternative pathway of complement and linked with the C3b activity. Two

patients had low C3 and factor B levels, clearly indicating a dysfunctional

regulation of the CAP. Although obtained in a very small number of sub-

jects, these findings suggest that an abnormal genetic control of the CAPmay

be a risk factor for the occurrence of HELLP syndrome and open new fields

of investigation.

96 BENEDETTO ET AL.

Previous reports indicate that HELLP syndrome appears in a more severe

form in patients’ carrier of antiphospholipid antibodies, and in those patients

it may be refractory to standard treatment leading more frequently to the

development of hepatic infarcts [89–91]. The role of the complement system

in antiphospholipid antibodies syndrome (APS) has been investigated in

murine models [92]. Passive transfer of human antiphospholipid antibodies

(aPL) resulted in complement activation and generation of split products

that induced placental injury and led to fetal loss/growth restriction [82,93].

The complement activation by aPL in other vascular areas may cause inflam-

mation and thrombosis. Mice deficient in C3 or C5 are less susceptible to

aPL-induced thrombosis and endothelial cell activation. Inhibiting C5 acti-

vation prevents aPL-induced thrombocytopenia, and mice treated with inhi-

bitors of complement activation are protected from fetal loss [92].

The complement and coagulation system are linked through both direct

and indirect interactions, and the vascular endothelium plays a key role in

this interaction. The complement increases blood clotting properties. C3a

and C5a induce platelet activation and aggregation, upregulation in PAI-1,

and tissue factor expression [94,95]. Complement also has thrombogenic

properties by decreasing protein S activity [96]. C3a and C5a contribute to

the regulation of the cytokine response, modulating production and secre-

tion of TNF-a and IL-6 by macrophages [95], which may enhance TF

expression and platelet production and activity. A damage of the endotheli-

al cell lining may activate the complement system and clotting, and the

thrombotic tendency of each organ varies depending on the local anticoag-

ulant and procoagulant characteristics. This may explain the tissue specific-

ity of the liver for HELLP syndrome, but the role of complement activation

in the pathogenesis of HELLP remains to be established.

The coagulation system seems to play a key role in HELLP syndrome.

Microthrombi formation, due to endothelial dysfunction and probably to

complement system activation, is essential in determining the clinical features

of the disease. The fibrinolytic system is necessary for clot dissolution, and in

a recent study, Guven et al. investigated the role of fibrinolytic and antifi-

brinolytic activities in the pathophysiology of HELLP syndrome [97]. They

measured the levels of plasma tissue-type plasminogen activator (tPA),

thrombin-activatable fibrinolysis inhibitor (TAFI), PAI-1, thrombin–

antithrombin complex (TAT), and thrombomodulin (TM) in normal preg-

nant women, nonpregnant women, and women with HELLP syndrome.

Compared to the control groups, the mean tPA, PAI-1, TAFI, TAT, and

TM levels were significantly increased in HELLP patients, suggesting an

involvement of fibrinolytic system in HELLP, possibly as a response to the

activation of coagulation system.

BIOCHEMISTRY OF HELLP SYNDROME 97

Moreover, components of fibrinolytic system may be involved in the

control of trophoblast invasion and spiral artery remodeling at the time of

placentation. It is known that MMPs and their specific inhibitors are

involved in this process [98]. The invasive behavior of trophoblast cells

correlates with MMP-9 expression, and tissue inhibitor of MMPs (TIMPs)

inhibits their invasive capacity. MMP-2 and MMP-9 cooperate closely with

the urokinase-type plasminogen activator (uPA) system, mutually activating

their components [98]. Synergism of these proteolytic factors in placental

development has been shown in animal experiments [99]. PAI-1, expressed in

villous and extravillous trophoblasts, as well in maternal decidual tissue, is

suggested to be closely involved in control of trophoblast invasion [100].

Recently, Pildner von Steinburg et al. observed that in patients with HELLP

syndrome, mRNA placental expression of MMP-2 and TIMP-2 was de-

creased, and mRNA expression of MMP-9 and uPA receptor was undetect-

able [101]. These findings, although preliminary, suggest a decrease in matrix

remodeling in placentae from patients with HELLP syndrome.

Preeclampsia is characterized by an imbalance between two cyclooxygen-

ase metabolites of arachidonic acid, thromboxane, and prostacyclin (PGI2)

that favors thromboxane. Because of the biologic actions of these two eico-

sanoids, this imbalance might explain major clinical symptoms of preeclamp-

sia, such as hypertension, platelet aggregation, and reduced uteroplacental

blood flow. In thematernal circulation, this imbalance is primarilymanifested

by decreased production of PGI2 by endothelial cells. Platelet thromboxane

synthesis is only increased in severe preeclampsia. In the placenta and leuko-

cytes, the imbalance is exacerbated by increased production of thromboxane

coupled with decreased production of PGI2 in both mild and severe pre-

eclampsia. Longitudinal measurements of urinary metabolites of thrombox-

ane and PGI2 reveal that the thromboxane/PGI2 imbalance predates the onset

of clinical symptoms of preeclampsia. The imbalance between thromboxane

and PGI2 is most likely caused by oxidative stress, which is manifest in

preeclampsia by increased lipid peroxidation and decreased antioxidant pro-

tection. Oxidative stress may drive this imbalance because lipid peroxides

activate the cyclooxygenase enzyme to increase thromboxane synthesis, but at

the same time they inhibit PGI2 synthase to decrease PGI2 synthesis [102].

Furthermore, the endothelial cell dysfunction typical of preeclampsia leads to

alterations in the release of vasodilator substances such as nitric oxide (NO),

PGI2, and endothelium-derived hyperpolarizing factor, and thereby reduc-

tions of the NO-cGMP, PGI2-cAMP, and hyperpolarizing factor vascular

relaxation pathways. These alterations may also increase the release of or the

vascular reactivity to endothelium-derived contracting factors such as

endothelin, thromboxane, and angiotensin II. These contracting factors

could increase intracellular Ca2þ concentrations ([Ca2þ]i) and stimulate

98 BENEDETTO ET AL.

Ca2þ-dependent contraction pathways in vascular smooth muscle. The

contracting factors could also increase the activity of vascular protein

kinases such as protein kinase C, leading to increased myofilament force

sensitivity to [Ca2þ]i and enhancement of smooth muscle contraction. The

decreased endothelium-dependent mechanisms of vascular relaxation and

the enhanced mechanisms of vascular smooth muscle contraction represent

plausible causes of the increased vascular resistance and arterial pressure

associated with preeclampsia. It is reasonable to think that these mechan-

isms are involved in HELLP syndrome, but they are not specific features of

HELLP [46].

6. Conclusion

Although the cause of tissue injury in HELLP syndrome is multifactorial,

a key role for prominent inflammatory response to abnormal placentation

may be suggested. Maternal vascular endothelium activation, complement

system defective regulation, and coagulation system activation are important

features of the disease. HELLP is categorized as a gestational hypertensive

disorder and seen as the more severe variant of preeclampsia. However,

several reports suggest that it may be a separate disease entity: differences

in placental genes expression and maternal polymorphic alleles involved in

inflammation responses confirm this hypothesis. Further studies are needed

to explain placental-induced disease, as in preeclampsia, since it involves a

more acute and predominant inflammatory process that typically targets the

liver with greater activation of the coagulation system. This occurs during a

disordered immunologic process and may be due to a genetic predisposition

in the particular involvement of the liver in HELLP syndrome, the prominent

inflammatory response, and the sensitivity to glucocorticoids.

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