Some Recent Insights into the Prothrombogenic Mechanisms of Antiphospholipid Antibodies

Abstract

Antiphospholipid antibodies (aPL) are a heterogeneous group of autoantibodies, detected in the sera of patients with both autoimmune and various non-autoimmune diseases. They are also detected in subjects with no overt underlying disease - the primary antiphosphilipid syndrome (PAPS). High titers of APL are associated with arterial and venous thrombosis, recurrent fetal loss and thrombocytopenia. There have been many suggestions explaining the potential mechanisms of the procoagulant effect of aPL. These include endothelial cell (EC) activation; increased adhesion molecule expression; inhibition of EC prostacyclin release, increased leucocyte adhesion to EC, downregulation of thrombomodulin expression. APL induce the procoagulant activity of monocytes via increased tissue factor expression and directly stimulate platelet hyperactivity with resultant production of enhanced amounts of the proaggregatory molecule of TXA(2). In vitro studies show that prepro-endothelin-1 mRNA is induced by human monoclonal anticardiolipin antibodies and this might contribute to vasospasm, and, ultimately, to arterial occlusion. The hypercoagulable state in APS patients is associated with alterations in the protein C/S pathway. It is suggested that aPL may impair the protein C anticoagulant system. Acquired protein C and protein S deficiency is described in patients with APS. Beta2- glycoprotein I, (Beta2-GPI) a natural anticoagulant, is involved in the regulation of protein S anticoagulant activity by preventing the binding of protein S to C4b-binding protein. APL were shown to inhibit this effect of Beta2- GP I. As the group of aPL is very heterogeneous, it is unlikely that a single mechanism is responsible for the thrombogenic activity of all aPLs associated with thrombosis.

Full text

Current Medicinal Chemistry, 2007, 14, 811-826 811
Some Recent Insights into the Prothrombogenic Mechanisms of
Antiphospholipid Antibodies
Maria Todorova*
,1
and Marta Baleva
2
1
Department of Pathophysiology, Medical University, Sofia, Bulgaria
2
Clinic of Allergology, Alexandrov’s Hospital, Medical University, Sofia, Bulgaria
Abstract: Antiphospholipid antibodies (aPL) are a heterogeneous group of autoantibodies, detected in the sera
of patients with both autoimmune and various non-autoimmune diseases. They are also detected in subjects
with no overt underlying disease – the primary antiphosphilipid syndrome (PAPS). High titers of APL are
associated with arterial and venous thrombosis, recurrent fetal loss and thrombocytopenia. There have been
many suggestions explaining the potential mechanisms of the procoagulant effect of aPL. These include
endothelial cell (EC) activation; increased adhesion molecule expression; inhibition of EC prostacyclin
release, increased leucocyte adhesion to EC, downregulation of thrombomodulin expression. APL induce the
procoagulant activity of monocytes via increased tissue factor expression and directly stimulate platelet
hyperactivity with resultant production of enhanced amounts of the proaggregatory molecule of TXA
2.
In vitro
studies show that prepro-endothelin-1 mRNA is induced by human monoclonal anticardiolipin antibodies and
this might contribute to vasospasm, and, ultimately, to arterial occlusion. The hypercoagulable state in APS
patients is associated with alterations in the protein C/S pathway. It is suggested that aPL may impair the
protein C anticoagulant system. Acquired protein C and protein S deficiency is described in patients with APS.
Beta2- glycoprotein I, (Beta2-GPI) a natural anticoagulant, is involved in the regulation of protein S
anticoagulant activity by preventing the binding of protein S to C4b-binding protein. APL were shown to
inhibit this effect of Beta2- GP I. As the group of aPL is very heterogeneous, it is unlikely that a single
mechanism is responsible for the thrombogenic activity of all aPLs associated with thrombosis.
Keywords: Antiphospholipid antibodies, Thrombosis, Procoagulant mechanisms
1. INTRODUCTION The growing interest in aPL over more than two decades
is attributed to their potential clinical significance, as it was
found that the presence of circulating aPL is associated with
anticoagulant effects in vitro, and, paradoxally with a
prothombotic state in vivo [1, 3, 5, 7].
Antiphospholipid antibodies (aPL) are a heterogeneous
group of acquired autoantibodies directed against negatively
charged phospholipids or phospholipids-binding proteins.
[1, 2]. APL are divided in two groups depending on the
method used for their determination. One group, the lupus
anticoagulant (LAC) antibodies, includes immunoglobulins
which inhibit phospholipids-dependent coagulation tests [3,
4]. LAC antibodies slow the rate of thrombin generation,
and therefore clot formation in vitro [5]. Thus, it is currently
believed that LAC blocks the in vitro assembly and activity
of the Xa-Va-Ca
++
- phospholipid complex (prothrombinase)
which is required for the conversion of prothrombin to
thrombin [6]. These antibodies show high affinity for
anionic phospholipids and very low affinity to neutral
phospholipids and DNA [4]. The other group includes
autoantibodies which meet the classical understanding of
antiphospholipid antibodies and is characterized by the
ability to bind to phospholipids in ELISA assays.
Cardiolipin (CL) is the phospholipid most often used in
these assays, and the detected antibodies are referred to as
anticardiolipin antibodies (aCL), but the phospholipids may
be also phosphatidylserine (PS) phosphatidylinositol (PI),
phosphatidylethanolamine, or phosphatidylcholine (PC) [4].
Subsequent studies showed close relationship between these
autoantibodies and LAC, both recognizing similar antigen
determinants, part of the coagulation cascade.
In 1990 was revealed that the phospholipid-binding
protein beta2-glycoprotein I (β2GPI) is required for the
binding of aCL in solid-phase immunoassay [8-10]. Later
other phospholipid-binding proteins as prothrombin,
annexin V, proteins S and C were also implied as
phospholipid cofactors [11,2]. The presence of antibodies to
prothrombin was first demonstrated by Bajaj et al. [12] in
patients with LAC- hypoprothrombinemia syndrome. Later
some affinity purified antiprothrombin antibodies were
found to have LAC activity [13]. An investigation on 357
patients with primary and secondary antiphospholipid
syndrome (APS) revealed that antibodies to both pure and
PS-complexed prothrombin are significantly associated with
lupus anticoagulant activity but only antibodies to pure
prothrombin display relationship to clinical manifestations
of APS [14]. Experimental studies proved that
antiprothrombin antibodies induce thrombosis and other
clinical manifestations of APS including thrombocytopenia,
and increased fetal resorption rate [15]. IgG antiprothrombin
antibodies are considered a significant marker of deep vein
thrombosis (DVT) and pulmonary embolism (PE) [16].
However, β2GPI is considered the major target for
antiphospholipid antibodies in patients with APS and it is
now generally accepted that anti- β2GPI antibodies are not
only a physiological marker, but that they are involved in
the pathophysiology of APS [17, 18]. Despite the increasing
*Address correspondence to this author at the Department of
Pathophysiology, Medical University, Georgi Sofiiski str., 1, 1431, Sofia,
Bulgaria; E-mail: todorovamariabg@yahoo.com
0929-8673/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.
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812 Current Medicinal Chemistry, 2007, Vol. 14, No. 7 Todorova and Baleva
interest in β2GPI and the extensive studies during the last
decade, its biological role is not fully understood. β2GPI is
a multifunctional plasma protein involved in many
anticoagulant/procoagulant pathways and physiological
reactions It acts as an anticoagulant by inhibiting the
activation of factors X and XII and thrombin generation [17,
19]. At the same time β2GPI exhibits procoagulant activity
by inhibiting both protein C activation, and the effect of
activated protein C on activated Factor V. When evaluating
the association of aPL with the clinical manifestations in
patients with systemic lupus erythematosus (SLE) it was
found that anti- β2GPI have a strong predictive value for
APS and association with arterial thromboses compared with
venous thromboses [20]. The presence of IgG anti- β2GPI in
patients with LAC and/or aCL predicts higher risk of
thromboembolic effects [21]. The complex anti- β2GPI/
β2GPI can activate platelets via the GPIalpha receptor, and is
supposed to contribute to thrombosis in APS [22].
Forastiero et al. [23] found impaired protein Z/protein Z
inhibitor system in patients with anti-β2GPI aPL and
assumed that this might increase the thrombogenic risk in
APS, as protein Z is considered to have anticoagulant
effects. The role and the opposing effects of β2GPI in
coagulation are summarized by Atsumi et al. [17]. The
structure and the mode of interaction of β2GPI with
phospholipids and aPL antibodies will be discussed further
in more details.
of APS requests persistent presence of medium to high
levels of aCL (IgG or IgM isotype), presence of LAC or
both [28]. In general, antibodies causing LAC are more
specific for APS, whereas aCL antibodies are more sensitive
[7]. The specificity of aCL for APS increases with titre and
is higher for IgG than for IgM isotype [4]. Except patients
with SLE, APS may affect patients with other related
autoimmune diseases, including Sjogren’s syndrome,
systemic sclerosis, Behçet syndrome, and infectious diseases
[5]. Unlike other thrombophilias, APS may be associated
with multiple other manifestations as livedo reticularis,
autoimmune thrombocytopenia, chorea, heart valve
abnormalities [5, 27].
Some patients, without other underlying disorders and
with uncomplicated history of thrombosis and recurrent
abortions were found to have raised aPL levels [29]. These
subjects were regarded as having a “primary
antiphospholipid syndrome” (PAPS) [26]. The most
frequently detected subgroups of aPL antibodies are LAC,
aCL antibodies, and anti-beta2 glycoprotein I (β2GPI)
antibodies [4]. During the last decade the nature and
pathophysiology of APS have been extensively reviewed [2,
4, 5, 7, 27].
Many pathologic states are associated with a great variety
of clinical manifestations in the presence of aPL [3, 6, 30-
37].
The MEDLINE search of the literature from 1988 to
2000 showed that the anticardiolipin titre correlated with the
odds ratio of thrombosis. The survey of the data showed that
the detection of LAC and, possibly of IgG aCL at medium
to high titers helps to identify patients at risk for thrombosis
[3].
In this review, we made an attempt to focus on some
basic pathways through which aPL antibodies exert their
thrombogenic effects.
2. ANTIPHOSPHOLIPID SYNDROME: ANTIPHOS-
PHOLIPID ANTIBODIES-CLINICAL EVIDENCE
FOR THROMBOSIS
The most common clinical implications, associated with
aPL are presented in Table 1.
The antiphospholipid (Hughes) syndrome (APS) was
described two decades ago as a distinct clinical entity [24,
25]. APS was first observed in patients with SLE, when it
was recognized that the presence of aPL as LAC and aCL
was associated with thrombosis, recurrent fetal loss and
thrombocytopenia [4, 7, 26, 27]. By definition, a diagnosis
2.1. Venous Thrombosis and Pulmonary Embolism
In 1986 R. Asherson [38] reported that the presence of
LAC and aCL is associated with recurrent venous
thrombosis. Deep vein thrombosis (DVT) of the legs is
Table 1. Clinical Implications and Manifestations of Antiphospholipid Antibodies
Clinical implications of aPL antibodies Manifestations of aPL antibodies
• SLE and other connective tissue disorders
• Primary APS
• Infectious diseases
• Lymphoproliferative diseases
• Drug induced aPL antibodies
• Absence of underlying disease
• Venous thromboembolic complications
• Arterial thromboses of macro- and microvessels
• Recurrent fetal loss
• Thrombocytopenia
• Unusual manifestations as:
-autoimmune thrombocytopenia
-autoimmune haemolytic anemia
-livedo reticularis
-heart valve disease
-skin ulcerations and necrosis
-blue toe syndrome, etc.
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Prothrombogenic Mechanisms of Antiphospholipid Antibodies Current Medicinal Chemistry, 2007, Vol. 14, No. 7 813
Fig. (1). Schematic representation of the interactions between phospholipids, β2GPI, and anti-β2GPI antibodies. A. Flat orientation of
β2GPI to the surface of the lipid layer. B. Upright orientation of β2GPI. C. Nicked β2GPI loses the property to bind to phospholipids.
often complicated with pulmonary embolism (PE). DVT/PE
is a frequent finding in patients with elevated levels of aPL
antibodies with 65% of them having IgG aCL [39]. Other
sites affected are the renal veins, extending from the inferior
vena cava, hepatic veins, associated with veno-occlusive
disease or a Budd-Chiari syndrome [5, 38]. DVT with LAC
and elevated levels of aCL were detected in 3 children aged
10-14 years [40]. In many patients early features are
symptoms compatible with pulmonary embolism and in one
of the cases these symptoms clearly preceded the recognition
of thrombosis [41]. Examination of patients, positive for
LAC and/or aCL for prevalence of DVT in the lower limbs
and the pelvic region and PE showed that DVT was detected
in 32%, and LAC
+
/aCL
+
DVT reached 53%. The prevalence
of PE was higher in patients positive for aCL [42]. A
correlation was found between the presence of anticardiolipin
antibodies and recurrent superficial thrombophlebitis [43].
Studies on two large cohorts of patients with both primary
and associated with SLE APS showed an association with
aPL and the coexistence of two or more complications [44,
45]. Thus were constructed the preliminary criteria for the
classification of APS in SLE, and with some modifications,
of primary APS [44]. A large study on 1000 patients
allowed to ascertain the incidence of different complications
and to find an association with SLE, the patient’s sex, the
patient’s age at disease onset, that can modify the disease
expression and the specific subsets of APS [45].
2.2. Arterial Occlusions
Occlusions of arteries of different caliber and site have
been reported in patients with aPL antibodies. They include
occlusions of the retinal artery; occlusions of the axillary
artery, giving rise to the aortic arch syndrome, mesenteric
artery occlusions with resultant bowel infarction [5, 38, 46].
Most commonly affected by arterial thrombosis are the
arteries of the central nervous system (CNS), and coronary
arteries.
2.2.1. Central Nervous System Manifestations
In 1986 Hughes et al. [47] proposed the principal
nervous system manifestations of APS. In a cohort of 162
patients with recent (<6 months) thrombo-embolic events
cerebro-vascular infarction affected 82 patients and in 56% of
them were found IgM aCL antibodies [39]. Thromboses,
occluding large cerebral arteries with consequent transient
ischemic attacks (TIA) or stroke are common in these
subjects. Patients without clinical or laboratory evidence of
SLE and with persistently elevated titres of circulating aCL
antibodies were found to have multiple cerebral infarctions
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814 Current Medicinal Chemistry, 2007, Vol. 14, No. 7 Todorova and Baleva
and dementia [48]. Ischemic stroke associated with aCL was
detected also in patients without SLE and LAC [49]. IgG
aCL antibodies, particularly β2GPI - dependent are
considered an important predictor of future stroke and
myocardial infarction (MI) in men [50]. Younger patients
without identifiable risk factors for atherosclerosis presenting
with stroke, are more likely to have an underlying APS as
an etiology, while no such association was observed for MI
patients [51]. A large study, comprising 524 hospitalized
acute stroke patients and 1020 community controls enrolled
in the Minorities Risk Factors and Stroke Study
demonstrated that aCL is an independent stroke risk factor.
IgG, and for the first time IgM aCL were each shown to be
associated with increased stroke risk. Moreover, the
prevalence of these antibodies and the stroke risk associated
appear greater than previously reported [52]. The results of a
population-based case-control study examining aPL
antibodies (aCL and LAC) in 180 cases and 340 controls
included in the Stroke prevention in Young Women Study
showed that the relative odds of stroke for women with
aCLs of any isotype or LAC was 1.87 (p=0.027) after
adjustment for age, current cigarette smoking, hypertension,
diabetes, angina, ethnicity and body mass index. According
to the authors these results support the importance of aPL
antibodies as an independent risk factor for stroke in young
women [53]. Investigation of the incidence of aPLs in 10
children with acute cerebral infarction showed that 7 of them
had aCL [54]. Furthermore, Katsarou et al. [55] reported on
a 20-month-old boy who had an ischemic stroke, secondary
to antiphospholipid syndrome with high titres of IgG anti-
β2GPI (at normal range 0-100 U; the first measurement of
the patient was 132 U, and the second value 6 weeks later
was 350U). This is the first case report of childhood
ischemic stroke with only anti-β2GPI, but no antibodies,
detectable in standard antiphospholipid assays.
determination of IgM may be a useful marker to discriminate
ectopic pregnancies with autoimmune pathogenesis from
those caused by other factors [71]. A study on 450 pregnant
women with a history of one or more spontaneous abortions
revealed that 72 (16%) of them were strongly positive for
IgG aCL [72]. Recently other aPL antibodies are supposed
to have clinical relevance in women with recurrent abortion
as well. A study enrolling 155 patients with three or more
recurrent pregnancy losses showed that aCL were detected in
40%, and 19% were positive for anti-PS IgG [73]. Others
suggest that anti-phosphatidylethanolamine antibodies of
IgG and IgM isotype found in patients with recurrent
abortions may be a risk factor for early and mid-to-late
pregnancy loss(es) [74]. The pathogenesis of recurrent
abortions due to aPL antibodies implies thromboses in the
placental vessels with decreased placental perfusion and
subsequent infarction, aPL-mediated inhibition of
trophoblastic invasion and vasculopathy [75, 76].
Experimental data confirmed the pathogenic role of aPL
antibodies. Monoclonal aCL antibodies administered to
pregnant mice lead to increased intrauterine fetal death due to
placental infarction [77, 78].
2.3. Unusual Manifestations
Although the classical features of APS comprise venous
and arterial thrombosis, recurrent fetal loss and
thrombocytopenia, some unusual clinical manifestations are
established in subjects with aPL antibodies. These include
livedo reticularis, Sneddon’s syndrome, chorea, adult
respiratory distress syndrome (ARDS), avascular necrosis of
the bones, renal thrombotic microangiopathy [5, 79, 80],
psychosis [81]. IgG aCL antibodies are present in 25% of
the patients with myasthenia gravis [82]. Additionally,
hemolytic anemia is relatively common in patients with
APS [79]. Patients with APS associated with SLE have a
higher prevalence of hemolytic anemia [83], and there is a
correlation between aPL of IgM isotype and the hemolytic
anemia [84].
2.2.2. Cardiovascular Disease (CVD)
Elevated levels of aPL antibodies are detected in patients
with stable and unstable angina and cases of MI, with an
association between the titres of aPL and CVD [56-61]. An
interesting study of survivors of myocardial infarction
revealed that aCL antibodies are common in young (under
45 y) postinfarction patients and represent a maker of high
risk for recurrent cardiovascular events. Eight of the 13
patients with raised aCLs experienced additional
cardiovascular events during a follow-up of 36-64 months
after the first myocardial infarction. They had aCL titres of 5
times the mean for voluntary blood donors [57]. Prospective
studies of middle-aged men revealed that elevated levels of
aCLs and antibodies against oxidatively modified LDL
predict MI [60, 61]. According to M. Petri [62] both LAC
and aCL antibodies are predictive of later venous or arterial
thrombosis, and myocardial infarction occurs significantly
more often in those with LAC. The incidence of anti-β2GPI
antibodies in patients with acute coronary syndrome is
significantly higher (14.4%) in comparison to control
healthy subjects [63]. IgG aCL are common in patients with
peripheral artery disease and are associated with an increased
risk of cardiovascular and overall mortality [64].
This characterizes the APS as a multisystem autoimmune
disease with large variety of clinical manifestations.
2.4. Drug Induced aPL
Both early and recent studies confirm the induction of
aCL antibodies by drugs. In 1989 Asherson et al. [36]
reported for the induction of a lupus-like syndrome with
aCL antibodies by procainamid. Young females with
presence of aCL developed thromboembolic disease while on
oral contraceptive therapy [85, 86]. Data suggest that there is
a high incidence of aCL and LAC in patients with psychotic
diseases and neuroleptic drugs increased the incidence [37,
87]. Larsson et al. [88] established renal insufficiency and
high titers of aCL in a patient while on thiazide. Four
months after withdrawal the autoantibodies disappeared and
the progression of renal insufficiency ceased. Drug induced
aCL usually are not associated with thrombotic events, even
if IgG aCL are predominant [37]. However, in patients with
SLE and circulating aCL, the administration of
contraceptives and hormone replacement therapy (HRT) is
associated with increased risk of thrombosis [89-91]. Data
2.2.3. Recurrent Fetal Loss
APL antibodies are a common finding in patients with
recurrent fetal loss(es) [65-71]. Moreover, data show that the
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Prothrombogenic Mechanisms of Antiphospholipid Antibodies Current Medicinal Chemistry, 2007, Vol. 14, No. 7 815
concerning the induction of aCL by sex hormones in normal
subjects are scarce. The first experimental study was made
by Ahmed et al. in 1993 [92] who demonstrated the
induction of aPL antibodies in normal mice by estrogen.
The estrogen-treated mice also had antibodies directed
against other membrane phospholipids including PI, PC, PS
and phosphatidylethanolamine. Later the same authors found
that the estrogen-induced aPLs were of IgM and IgG, but not
of IgA isotype. The expression of these antibodies persisted
for months after the exposure to exogenous estrogen has
been terminated [93]. Our data in healthy postmenopausal
women on HRT showed a significant transitory elevation of
IgM, but not of IgG aCL [94]. Treatment of healthy women
after the menopause with tibolone, a synthetic steroid with
weak estrogenic, androgenic and progestagenic properties
affected the levels of aCL in a similar pattern, but the
increase of IgM was not statistically significant [95].
In 1990 three research groups independently reported that
affinity-purified aCL antibodies were not directed to
cardiolipin itself, but to a cofactor β2GPI, a plasma protein
which binds to cardiolipin [8-10]. Thus aCL antibodies were
characterized as cofactor-dependent or cofactor-independent
according to the need of β2GPI for the binding to
cardiolipin. In patients with autoimmune diseases and APS
aCL antibodies require β2GPI to bind to cardiolipin and are
associated with thrombosis, fetal loss, thrombocytopenia or
other clinical manifestations, while aCL antibodies, induced
by infections usually do not require β2GPI and are generally
considered non-pathogenic [9,10]. Some data however show
that aCL antibodies, induced by infections also can be
associated with clinical manifestation [101]. On the other
hand, different infections can trigger thrombotic events in
patients with APS, including the potentially lethal subset
termed catastrophic APS [102].
3. ANTIPHOSPHOLIPID ANTIBODIES – ISOTYPE
DISTRIBUTION AND RELATION TO 2GPI
4. 2GPI – STRUCTURE, CONFORMATIONAL
STUDIES, ACTIVITY, AND BIOLOGICAL
IMPLICATIONS
In 1987 Gharavi et al. [96] first determined the isotype
distribution of aCL antibodies in patients with one or more
of the aPL-associated clinical complications – thrombosis,
fetal loss, thrombocytopenia. Twelve of 40 patients had IgG,
IgM, and IgA aCls, 10 patients had IgG and IgM, 5 patients
had IgG and IgA, and 3 patients had IgM and IgA aCL
antibodies. Consequent studies supported the finding that
IgG, followed by IgM aCL are the most frequently
distributed and clinically relevant aCL antibodies found in
patients with APS [39, 52].
In 1961 Schultze et al. [103] first described β2GPI, a
perchloric soluble plasma protein with an unknown function.
Recently β2GPI is thought to be the key player in APS and
much work has been performed to elucidate its structure,
physiological role, mode of interaction with negatively
charged phospholipids and aPL antibodies and its role in the
pathophysiology of APS [5, 9, 10, 17 ].
4.1. Structure
Anticardiolipin antibody isotypes and titre are two
important issues. The pathogenicity of aCL of different
isotypes is not well defined yet. Most former studies
investigated predominantly IgG autoantibodies and did not
distinguish between isotypes. Significant associations with
thrombotic events were found for IgG aCL, especially for
titers above 33-40 units [53, 55]. An international consensus
established that aCL antibodies at medium to high titre are a
criterion of definite antiphospholipid syndrome [28]. Recent
data however point that isotypes IgM [52] and IgA [31] also
have clinical significance in patients with SLE and primary
APS. Some data indicate, that IgG and IgM aCL antibodies
seem to define different clinical subsets of patients with
thrombosis with IgG being most prevalent in the group
having DVT/PE, and IgM being found primarily among
cerebro-vascular infarction patients [39].
β2GPI, known also as apolipoprotein H (Apo H) is a
glycoprotein normally found in plasma either as a free
protein or associated with lipoproteins. About 16% of
plasma β2GPI is found with chylomicrons and VLDL, 2%
with LDL, 17% with HDL and the remainder (65%) in the
1.21 density infranatant [104]. As β2GPI is isolated on
plasma lipoproteins with high affinity for triglyceride (TG)-
rich lipoprotein particles and activates lipoprotein lipase in
vitro, Lee et al. [105] postulated that β2GPI satisfies all the
criteria to be classified as an apolipoprotein and designated it
as apo-H.
The plasma concentration of β2GPI is approximately
200µg/ml. It is a 54 kDa single-chain polypeptide
consisting of 326 amino acid residues with five
oligosaccharide N-glycosylation sites [106-108]. The amino
acid sequence is highly conserved (>80%) in different
species as human, bovine and mouse, indicating important
biological function [106, 108-111].
IgA aCL and IgA anti-β2GPI antibodies are common in
SLE and are associated with thrombosis and
thrombocytopenia [97]. Anti-β2GPI antibodies of IgA
isotype might be most relevant for the onset and outcome of
acute coronary syndrome [63]. Infections may have a greater
role in the incidence of IgA aCL than IgA anti-β2GPI
antibodies [98].
β2GPI consists of five homologous motifs (short
consensus repeat domains, SCR) of approximately 60 amino
acids each with two internal disulfide bonds. Domains I-IV
are identical in structure, and the fifth domain contains a
positively charged lysine cluster and a hydrophobic extra C-
terminal loop [112]. The fifth domain consists of residues
244-326 and is stabilized by three internal disulfide bonds.
β2GPI is a member of the superfamily of proteins known
also as complement control proteins (CCP) or Sushi domain
superfamily, often involved in the protein-protein
interactions [108-113]. CCP domains are common in many
The incidence of circulating aPL antibodies shows
seasonal distribution [99] and can be influenced by age and
feeding pattern. Autoimmune-prone NZB/WF1 mice fed on
high fat diets had higher IgG aCL antibodies at the age of 3-
4 months, while IgM aCL did not differ compared with the
control group [100].
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816 Current Medicinal Chemistry, 2007, Vol. 14, No. 7 Todorova and Baleva
proteins (over 140) involved in the complement regulatory
system, such as complement factor H, CR1, C1, C2 and
C4BP and in blood coagulation as factor XIII [114, 115].
4.3. Nicked form of 2GPI
In 1993 Hunt et al. [123] suggested that the decreased
interaction of commercial β2GPI with cardiolipin can be
attributed to the fact, that the commercial preparation of
human β2GPI contained a nicked form of the protein in
domain V. The authors found two cleavage sites in domain
V of commercial human β2GPI. The major site, located at
Lys
317
-Thr
318
, and the other, minor site at Ala
314
-Phe
315
.
The nicked form of β2GPI is generated by different
proteases, found normally in human plasma. Factor Xa
produced the nicked form of domain V by cleavage of the
Lys
317
-Thr
318
bond and the nicked form lost the ability to
interact with CL, however the reaction was extremely slow
[124]. Later was established that plasmin more readily
produced the nicked form of β2GPI either by incubation of
purified β2GPI with plasmin or by activation of
plasminogen in plasma by urokinase (UK). The reduced
affinity of the nicked β2GPI to CL was attributed to the
conformational changes of domain V caused by the cleavage
of Lys
317
-Thr
318
[118].
In 1999 two groups of scientists described the crystal
structure of human β2GPI and the mechanism of interaction
with phospholipid surfaces, thus contributing to the
understanding of the pathophysiology of APS [116, 117].
The fifth domain of β2GPI has been implicated in membrane
binding. The positive charges on domain V interact with the
anionic phospholipid headgroups and the flexible loop Ser
311
-Lys
317
inserts into the lipid layer, thus anchoring the
protein molecule to the membrane [116]. The residues,
responsible for the unique function of domain V in
membrane binding are located on the aberrant non SCR-like
half of this domain. The phospholipid -binding property of
β2GPI is significantly attenuated by cleavage between
Lys
317
and Thr
318
in the domain V [118]. Schwarzenbacher
et al. gave some more details concerning the crystal structure
of β2GPI. The molecule has an elongated J-shape (like a fish
hook) with overall dimensions of 132x72x20 Å. In the
crystal structure the authors identified four N-linked
glycosylation sites: three of the glycans are located in
CCP3, and the fourth is part of CCP4. The functional role
of the carbohydrate moiety is not yet clear; however
glycosylation in vivo generally leads to inhomogeneity of
the protein and allows for the isolation of distinct isoforms
[117].
Increased levels of nicked-β2GPI are detected in different
pathologic states as disseminated intravascular coagulation
(DIC) [125], in patients with leukaemia with lupus
anticoagulant [126], ischemic stroke [127]. These conditions
are characterized by excessive thrombin generation and fibrin
turnover and the conversion of plasminogen to plasmin is a
key event in extrinsic fibrinolysis. In vivo, during thrombus
formation and thrombolysis β2GPI is cleaved by plasmin to
a nicked form. Yasuda et al. first demonstrated the role of
nicked - β2GPI in fibrinolysis. They revealed that the nicked
- β2GPI binds to the naturally circulating form of
plasminogen Glu-plasminogen, and suppresses plasmin
generation up to 70% in the presence of tissue plasminogen
activator, plasminogen, and fibrin. [127]. Thus, the nicked
form of β2GPI affects the extrinsic fibrinolysis via a
negative feedback pathway loop [128]. The levels of β2GPI
are reduced in patients with DIC and thrombosis [129],
probably due to the increased generation of nicked-β2GPI,
which is more readily cleared from the circulation compared
with native β2GPI. The detection of nicked-β2GPI in plasma
represents a sensitive marker of cerebral ischemic events
which allows to assume that the nicked form of β2GPI may
have a different physiological significance compared with the
native protein [17].
4.2. Domain V and Implication for Lipid Binding
Domain V is characterized by an unusual amino acid
composition, and provides an excellent counterpart for
electrostatic interactions with negatively charged amphiphilic
substances. This explains the fact that the integrity of the
disulfide bonds is crucial for the lipid-binding capacity of
β2GPI [112, 119, 120]. Moreover, a genetically determined
mutation where Trp
316
is substituted by Ser
316
completely
abolished the phospholipid-binding capacity of β2GPI [94,
102].
The biological function if β2GPI is ultimately dependent
on the interactions of the protein with lipid membranes
[121]. Wang et al. [121, 109] elucidated the mechanism of
insertion of Apo H into membranes during lipid-protein
interaction. The authors found that ApoH undergoes a
genuine conformational change as it binds to phospholipids
and this change can be characterised at the secondary
structure level. The secondary structure of ApoH is not
sensitive to pH change and is more probably dependent on
the dielectric features of the microenvironment. A
consequtive study of Wang et al. [122] revealed that human
ApoH may have various orientations when attached to lipid
layers. On neutral lipid layer ApoH has an upright
orientation, which is not sensitive to the phase state of the
lipid. On acidic lipid layer, however, Apo H may have two
forms of orientation. One is an upright orientation in the
liquid phase region, and the other is flat orientation on the
condensed domain region.
4.4. Binding of Anticardiolipin Antibodies to 2GPI
ACLs show heterogeneity in their binding to different
phospholipids [130, 131, 10]. For this reason the mode of
interaction of aCL, β2GPI and CL is an object of interest
and intensive studies. The knowledge of the epitope(s)
location for pathogenic aCLs would permit a more specific
therapeutic approach with agents, acting as toleragens for the
specific B cells, producing the autoantibodies [132, 133].
Different epitopes for aPL were identified, located on the
domains of β2GPI. Matsuura et al. [134] showed that aCL
antibodies may bind directly to β2GPI, coated on an
irradiated polystyrene surface in the absence of CL. The
authors suggested that β2GPI undergoes conformational
changes on interacting with an oxygenated solid phase
surface, exhibiting a new epitope, which is recognized by
These variations of spatial orientation of the molecule on
the lipid layer may relate to the variety of its physiological
and pathogenic functions [122].
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Prothrombogenic Mechanisms of Antiphospholipid Antibodies Current Medicinal Chemistry, 2007, Vol. 14, No. 7 817
aCL antibodies. This suggests that aCLs from APS patients
react to the complex β2GPI-CL in an aCL ELISA in a
similar way. These results allowed the authors to conclude
that the immune response could be addressed to lipid-bound
and structurally modified β2GPI, rather than to lipid or
β2GPI alone.
antithrombin III to accelerate thrombin inhibition; 3)
annexin V, which prevents binding of coagulation factors,
and 4) by release of tissue factor pathway inhibitor (TFPI),
which blocks the factor VIIa-TF-Xa complex [138].
Endothelial dysfunction results in a prothrombotic state, in
which increased production of tissue factor (TF),
plasminogen activator inhibitor (PAI), adhesion molecules,
endothelin-1 (ET-1) and von Willebrand factor (vWF), and
decreased production of thrombomodulin (TM) create a
prothrombotic surface on the vascular endothelium [139].
Some anti-β2GPI antibodies bind to domain I [135].
Further investigations on the mechanism of binding of aCL
to β2GPI established that the cryptic epitopes for aCL are
located on different domains - III, IV, and V with prevalence
of domain IV [106]. For the purpose were used mouse anti-
β2GPI monoclonal antibodies (moAb-cof 18, 20, 21, 22 and
23) and it was established that different moAb bind to
different epitopes. Thus cof18 bound only to intact β2GPI
and domain V, but not to their nicked forms, and recognised
a structure, related to both phospholipid binding and
plasmin-mediated cleavage. The structure analysis showed
that the cluster of amino acids which formed a cof-18’s
epitope stretched after the cleavage. In contrast, cofs 20, 21,
22, and 23 reacted with both intact and nicked β2GPI, with
epitopes located on domains III and IV. In ELISA using
β2GPI-adsorbed plates, binding of cof-23 to domain IV was
affected by treatment with plasmin, which indicated that
evidently the nicked form of β2GPI induced a
conformational change in domain IV, which reduced the
binding affinity of aCL antibodies to the protein [106]. Bas
de Laat et al. showed that thromboembolic complications
were best associated with antibodies directed against domain
I [135].
APL antibodies activate EC and increase their
procoagulant activity [138, 140]. Studies of Pierangeli et al.
[138] showed that aPL antibodies of IgG isotype isolated
from APS patients activate EC in vitro and in vivo. The
affinity purified aPL antibodies from patients with diverse
clinical features of APS increased adhesion of leukocytes to
EC in vivo, indicating their activation. The aPLs also
enhanced thrombus formation and delayed the time of
thrombus disappearance significantly. In vitro studies
showed that the same aPL antibodies significantly increased
the expression of VCAM-1 (2.3 to 4.4 fold) and the
expression of E-selectin (1.6 fold) on human umbilical vein
endothelial cells (HUVECs) after aPL exposure [138]. These
data are in agreement with previously published results from
the same group which showed that monoclonal and
polyclonal aPL antibodies isolated from APS patients with
thrombosis enhanced thrombus formation in vivo in a dose-
dependent fashion [141]. Similar effects exert murine
monoclonal aPL antibodies, having aPL and anti-β2GPI
activity. Mice, injected with monoclonal aPL showed a
significant increase in leukocyte sticking and produced larger
thrombi that persisted longer. Exposure to these aPLs
significantly increased surface expression of E-selectin,
ICAM-1 and VCAM-1. These data indicate that aPL
antibodies, induced by immunization with the
phospholipids-binding site of β2GPI are thrombogenic and
activate EC [140].
Yasuda et al. [136] identified and reported 2 Japanese
families with β2GPI deficiency due to a single mutation
deletion. Notwithstanding the multiple functions of β2GPI,
its genetic deficiency does not represent a risk factor for
coagulation disorders like thrombosis or excessive bleeding
in humans [137].
5. PATHOGENETIC MECHANISMS OF THROMBOSIS
In an in vivo experimental model was shown that aCL
antibodies of IgG, IgM and IgA isotype caused a significant
increase in mean thrombus area and a significant delay in
mean thrombus disappearance time [141]. Recent studies of
Pierangeli et al. [142] revealed that the activation of EC and
enhanced thrombosis by aPL, reported previously in vivo
[138, 140] are mediated by ICAM-1, P-selectin, or VCAM-
1. An experimental model with ICAM-1 and ICAM-1/P-
selectin – deficient mice, treated with affinity-purified aPL
antibodies showed that ICAM-1, P-selectin and VCAM-1
expression are important in thrombotic complications caused
by aPL antibodies in APS patients [142].
Regardless of the numerous data proving the
involvement of aPL antibodies in the clinical complications
of APS, the mechanism(s) of thrombosis in APS patients
are not completely clarified. On the one hand this is due to
the multiple factors involved in the processes of coagulation
and fibrinolysis, and on the other hand it might be due to
the great heterogenity of the antiphospholipid antibodies, as
isotype, specificity for phospholipids, and phospholipid-
binding plasma proteins. Different potential pathways
through which aPL antibodies promote thrombosis have
been proposed [4, 5, 46]. The mechanisms can be
provisorily divided into several subgroups.
Experimental data proved that some murine aCL, but not
anti-β2GPI antibodies are thrombogenic in vivo, suggesting
that the thrombogenic effect of aPL antibodies is not related
to their anti-β2GPI activity alone [143].
5.1. Cellular Mechanisms in the Pathophysiology of
Thrombosis in APS
In vitro studies demonstrated the activation of HUVECs
by IgG from patients with high-titre aCL antibodies. The
antibodies induced a 2.3–fold increase in monocyte
adhesion. Immunofluorescent microscopy showed that EC
incubated with patient IgG aCL antibodies expressed E-
selectin, VCAM-1, and ICAM-1 [144]. Different aPL
antibodies from the same patient were found to have
different pathogenicity [145].
5.1.1. Endothelial Cells
It is well known that the vascular endothelium plays an
important role in maintaining the anticoagulant surface of
blood vessels and the procoagulant/anticoagulant balance.
This is achieved through expression of 1) thrombomodulin
by activating protein C; 2) heparin sulfate by activating
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818 Current Medicinal Chemistry, 2007, Vol. 14, No. 7 Todorova and Baleva
Functional analysis showed that about 2/3 of patient-
derived IgG monoclonal aCLs are thrombogenic, and some
of them exert the effects through activating EC and
endothelial expression of adhesion molecules [146]. APL
antibodies induce significant increase in the transcription,
expression and activity of TF, and interleukin (IL)-6 and IL-
8 up-regulation in cultured HUVECs. The intracellular
events in aPL-mediated EC activation involve p38 mitogen-
activated protein kinase (MAPK) and nuclear factor (NF)-
kappa B [147]. The activation of NF-kappaB and the
increase in phosphorylation of p38MAPK in EC by aPL
antibodies could be reversed by specific inhibitors MG132
and SB203580, respectively [148].
5.1.2. Monocytes
The procoagulant activity of monocytes is associated
predominantly with cellular adhesion and the secretion of
tissue factor (TF), the main physiological initiator of blood
coagulation in vivo [162]. In the presence of phospholipids it
binds to factor VII/VIIa, which results in activation of
factors IX and X, and, finally, to thrombin formation.
During the last decade many studies indicated that aCL
activate monocytes [143, 163-166].
Murine monoclonal aCL IgG can induce a TF-like
procoagulant activity on monocytes [163]. Similar effects
had human monoclonal aCL by inducing procoagulant
acivity and upregulation of TF mRNA in monocytes.
Monocytes of patients with primary APS show increased TF
surface expression, which correlated with thrombogenic
events in the patients. The surface expression of TF on
monocytes was associated with IgG, but not with IgM aCL
antibodies or LAC [164]. Lackner et al. [165] found that
two human monoclonal IgG aPL antibodies were able to
induce tissue factor-like activity on human peripheral blood
monocytes. Neither antibody was dependent on β2GPI.
High levels of some endothelium-derived factors as ET-
1, TM, vWF and soluble E-selectin are associated with a
poor cardiovascular prognosis [149, 150].
In vitro human monoclonal aCL induced prepro-ET1
mRNA levels significantly more than did control
monoclonal antibody, lacking aCL activity. The plasma
levels of ET1 in APS patients were markedly increased and
both in vivo and in vitro results indicated that in APS
patients ET-1 might contribute to increased arterial tone,
vasospasm, and, ultimately to arterial occlusion [151].
Monocyte procoagulant activity (tissue factor production)
was significantly higher in SLE patients with LAC and/or
aCL in comparison to SLE patients without LAC/aCL and
to controls. This was strictly related to an increased
monocyte activation that could play an important role in the
occurrence of thrombotic events [166].
Thrombomodulin is an important membrane-bound
anticoagulant. Increased plasma levels of its soluble forms
may indicate loss of TM from the EC membrane and thus a
procoagulant state [152], and the decrease of soluble TM
levels may be considered favourable with respect to
cardiovascular risk [153]. In 30 children after ischemic stroke
were found increased levels of aCL, anti- β2GPI, and TM
compared with healthy controls [154]. Experimental data
showed that four of eighteen aCL antibodies, derived from
NZB/WF1 mouse reacted with rabbit TM and induced
down-regulation of TM on endothelial cells, followed by
induction of thrombosis [155]. Other studies, however,
indicate that although some monoclonal aCL bound to EC,
none of them reacted with TM. [156]. Evidently as aPL
antibodies in APS patients are heterogenous, the cross-
reactivity to TM of certain aCL is only one of the
mechanisms of aCL-induced thromboembolism.
Significant increase of TF expression was observed on
normal monocytes induced by three monoclonal IgM aCL
and 2 affinity-purified IgM aCL [167]. The accumulation of
TF mRNA was elevated in freshly isolated mononuclear
blood cells from patients with primary APS and was low or
absent in cells from normal controls [168]. The authors
however did not exclude the involvement of factors other
than aCL or LAC in inducing TF expression in APS
patients.
TF expression on monocytes is associated with high
levels of aCL in patients with APS and thrombosis [164].
Others do not find association between aCL titre and TF
activity in SLE patients which is attributed to adjusting for
other clinical factors as active arthritis, previous thrombosis,
and use of NSAID, which were found to significantly
modify TF activity [169].
In general, the biosynthesis of PgI
2
and TXA
2
is
increased in conditions with increased platelet turnover. In
patients with LAC is detected imbalance of the
thomboxane/prostacycline biosynthesis - platelet activation
and increased TXA
2
generation is associated with a failure of
the vessel wall to produce increased amounts of PgI
2
[157,
158].
Investigation of intracellular events showed that p38
MAPK phosphorylation, NF-kappa B translocation and TF
mRNA expression triggered by aCL/β2GPI were abolished
in the absence of β2GPI. Moreover, a specific p38MAPK
inhibitor SB 203580 decreased aCL/β2GPI induced TF
mRNA expression [170]. Thus, the p38MAPK pathway
plays an important role in the aPL-mediated activation not
only of EC but of monocytes as well. The proposed
mechanisms of cell activation induced by aPL are
comprehensively discussed by Yasuda et al. [171].
During the last decade several studies established
elevated values of vWF in patients with APS. According to
Ferro et al. [159] a prothrombotic state in patients with LE,
positive for aCL exists only when there is derangement of
EC, which the authors confirmed by finding increased levels
of vWF. Immunoglobulins from thrombosis prone patients
with SLE and APS stimulated the release of vWF antigen
[160]. Recently Levy et al. [161] found that the increased
platelet adhesion and aggregation in APS patients with
thrombotic events was associated with higher levels of vWF
antigen and ristocetin cofactor activity.
5.1.3. Platelets
Platelets (PLT) play a pivotal role in the control of
hemostasis. Phospholipase A2 releases arachidonic acid in
platelet membranes, which is converted into PGH
2
, that in
turn is metabolized to thromboxane A2 (TXA
2
). The process
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Prothrombogenic Mechanisms of Antiphospholipid Antibodies Current Medicinal Chemistry, 2007, Vol. 14, No. 7 819
of thrombus formation greatly depends on the concentration
of TXA
2
and prostacycline (PGI
2
), produced by EC in the
circulation. Platelets provide intrinsic platelet coagulation
proteins and simultaneously function as catalytic membrane
surfaces, leading to a significant enhancement of thrombin
formation [172]. Activated PLT adhere to subendothelial
components via adhesive proteins such as vWF. On
activation, the PLT membrane glycoprotein GPIIb-IIIa is
converted to a high-affinity receptor for fibrinogen and vWF,
and the binding with these molecules results in platelet
bridging and aggregation [172].
Normally, the binding of a protein to the cellular surface
is not enough to activate cells. The transduction of the
signal is usually accomplished through a specific receptor.
Platelet activation by thrombin is mediated by binding of
thrombin to GPIbα on the platelet surface before it can
activate the platelets via interactions with PAR-1 or PAR-4
[180]. The suggested receptor involved for platelets is
apoER2’. Apo ER2’ is a member of the LDL-receptor
superfamily (LDLR-family). LDLR belongs to the so-called
multiligand receptors with a broad specificity for structurally
and functionally dissimilar ligands [181, 182]. The finding
that apoER2’ can act as a receptor for β2GPI–antibody
complexes on platelets may further elucidate some aspects of
our understanding of APS [27]. Domain V of β2GPI
contains the binding site for the members of the LDLR-
family [183]. This interaction of β2GPI /apo ER2’ results in
phosphorylation of apo-ER2’, followed by phosphorylation
of p38MAPK and synthesis of thromboxane A2, which
mediates further activation of the platelets. These
assumptions were confirmed by Pierangeli et al. [148], who
found that aPL induce the production of TXB2 mainly
through activation of p38MAPK and subsequent
phosphorylation of cytosolic phospholipase A2.
Platelet activation and increased functional activity are
supposed to have a significant role in APS, particularly in
arterial thrombosis [4, 5]. Whether aPL antibodies activate
PLT in vivo is still a question of debate. In APS patients
circulating platelets are with increased functional activity,
but whether this state is a prerequisite or a result of
thrombosis is not clear. It was shown that human
monoclonal aCL increased the platelet interaction with
subendothelium under flow conditions [167]. Murine
monoclonal antibodies to β2GPI potentiate the platelet
aggregation response to some agonists [173]. However,
human IgG antibodies from patients with APS were found
to bind to platelets without induction of platelet activation
[174]. Robbins et al. [175] hypothesized that the complexes
aPL/β2GPI induce increased production of thromboxane
when interfering with platelets, previously activated by the
natural agonist thrombin. IgG aCL antibodies and plasma
aCL/β2GPI complexes were isolated from patients with
primary APS, high titres of IgG aCL and thrombotic
cerebrovascular disease. They found: 1) significantly
increased in vitro TXB2 production by platelets from
controls after incubation with aCL/β2GPI complexes; 2)
moderately increased TXB2 production by aCL alone; 3) no
increase in TXB2 production by β2GPI alone and 4).
significantly increased 11-dehydro-TXB2, a metabolite of
TXB2 production in vivo, in the urine of patients with APS,
compared with healthy controls [175]. These and other
similar data [176] suggest that aCL/β2GPI complexes play a
role in promoting platelets to produce thromboxane, which
could contribute to the prothrombogenic state in APS
patients. Moreover, a positive correlation was reported
between the levels of urinary 11 dehydro TXB2 and the
titres of anti-β2GPI antibodies in APS patients [177].
5.2. Humoral Mechanisms
Another basic pathway through which aPL antibodies
promote thrombosis implicates interference with and/or
modulation of the physiological activity of plasma proteins
involved in hemostasis.
5.2.1. Protein C/Protein S Anticoagulant Pathway
In 1989 Ruiz-Arguelles et al. [184] reported for a patient
with primary APS, high titre of IgG aCL, recurrent venous
thrombosis and pulmonary thromboembolism. Coagulation
studies revealed normal values of protein C antigen and
functional deficiency of protein C, that was considered
acquired. The authors attributed the deficiency to the
interaction of aCL antibodies with TM, as the activity of
aCL antibodies decreased in a dose-dependent fashion when
preincubated with increasing amounts of TM.
TM binds to thrombin, converting it from a procoagulant
to an anticoagulant, capable of activating protein C.
Activated protein C (APC), in turn, inhibits clotting by
proteolytic cleavage of factors Va and VIIIa. Protein C
requires as a cofactor protein S, synthesized by EC [172].
Arnout et al. [178] suggested that thrombosis in APS is
analogous to that in heparin-induced thrombocytopenia.
According to this hypothesis β2GPI binds to the surface of
activated platelets, which allows the antibody binding to the
surface-bound β2GPI and a subsequent platelet activation
through membrane Fcγ RII receptors. Similar process may
take place on the surface of the endothelial cells as well.
Both experimental and clinical studies suggest that aPL
antibodies promoting thrombosis interfere with the protein
C anticoagulant pathway. We found a strong association
between decreased protein C activity and the presence of aCL
and anti-β2GPI antibodies. The decreased protein C activity
depended on the antibody titre [185]. The two main steps of
interference affect either the activation of protein C through
thrombin/thrombomodulin, resulting in a decrease in
activated protein C (APC), or increased APC-resistance and
attenuated degradation of factors Va and VIIIa by the protein
C/S complex [11].
Recent studies elucidated the intracellular events in
platelet activation induced by aPL in the presence of low
doses of thrombin. APL antibodies in the presence of
subactivating doses of thrombin induce the production of
TXB2 mainly through the activation of p38MAPK and
subsequent phosphorylation of Ca
2+
- dependent cytosolic
phospholipase A2 (cPLA2). The pretreatment of the platelets
with SB203580, a p38MAPK inhibitor completely
abrogated the aPL-mediated enhanced aggregation of the
platelets [148, 179].
Macko et al. [186] performed a case-control study on 36
patients with acute brain infarction with preceding systemic
infections/inflammatory syndromes and hospitalized
nonstroke neurological patient controls. They found that the
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820 Current Medicinal Chemistry, 2007, Vol. 14, No. 7 Todorova and Baleva
mean circulating APC level was significantly lower in the
stroke group. C4b-binding protein (C4bBP) levels were
higher in the stroke group with antecendent infection within
1 week, and there was a trend toward higher plasma total
protein S antigen but free protein S levels were not
measured. Remarkably, they found a significant inverse
relationship between levels of circulating APC and aCL
titers in the stroke patients, suggesting that aCL antibodies
may have inhibitory effect on protein C activation in vivo. In
non SLE patients with DVT and presence of LAC, aCL and
anti-β2GPI antibodies, was found an association between
acquired APC resistance and objectively documented DVT.
Multivariate logistic analysis revealed that the presence of
anti-protein S antibodies was the most significant risk factor
for DVT. The authors suggested that acquired APC
resistance may reflect functional interference of the protein C
pathway by anti-protein S antibodies [187]. β2GPI–
dependent aPL antibodies exerted inhibitory effect on APC
and its cofactor protein S, on factor Va degradation,
inducing APC resistance [188]. Furthermore, antibodies to
β2GPI, circulating in patients with APS suppressed tissue
factor pathway inhibitor (TFPI)-dependent inhibition of TF-
induced coagulation, which resulted in an increased FXa
generation [189].
this effect was abolished by human monoclonal aCL. The
authors suggested that human monoclonal aCL increased the
affinity of C4bBP for protein S. The latter may represent a
target for aCL when combined with β2GPI and cardiolipin
which explains the acquired free protein S deficiency and the
risk of thrombosis in patients with aCL. Upon binding to
C4bBP protein S looses its cofactor activity for protein C
[196]. Thus the reported functional deficit of protein S can
be due to increased binding to C4bBP. C4bBP is present in
human plasma in two major forms, one consisting of 6 or 7
α- chains and the other containing α- chains and a 45 kD β-
chain. The binding site for protein S on C4bBP is localized
on the β-chain [197]. Examination of total, free, and bound
protein S and C4bBP showed that all C4bBP containing a
β-chain was in complex with protein S, indicating that the
concentration of free protein S is regulated by the
concentration of C4bBP containing a β-chain [198]. It is of
interest to note that during inflammation the increase of
C4bBP as an acute-phase protein is predominantly on the
account of the protein lacking a β-chain [199]. This is
considered a physiological mechanism to prevent depletion
of free protein S.
In a preliminary report Walker et al. [200] suggested that
β2GPI interferes with the binding of protein S to C4bBP.
Later Merril et al. [194] found that preincubation of protein
S with equimolar C4bBP decreased protein S function of
approximately 40%. Addition of excess β2GPI reversed the
inhibition of protein S by C4bBP. Four mouse monoclonal
antibodies to β2GPI were used to evaluate the specificity of
the β2GPI effect. One of the four antibodies to β2GPI
inhibited its modulation of protein S activity and its
interference with protein S binding to C4bBP. These data
allow to asume that some aPL antibodies directed to β2GPI
might additionally contribute to thrombosis by interference
with the protein C/S anticoagulant pathway through β2GPI.
Another study showed an inhibitory effect of β2GPI on the
activation of factor X. β2GPI-dependent aCL antibodies
induced hypercoagulation or thrombus by blocking this
inhibitory effect of β2GPI [201]. The suggested
interrelations of aPL antibodies, β2GPI, and the protein C/S
system is shown on Fig. (2).
Experimental data showed that murine monoclonal aCL
decrease the expression and internalization of TM attenuating
its anticoagulant activity [155]. In a cross-sectional study on
50 patients with SLE Tomas et al. examined the levels of
protein C, total and free protein S, antithrombin III (AT III),
and plasminogen in conjunction with the presence of LAC
and 3 types of aPL (anti-CL, anti-PS and anti-PI). They
found reduced protein S levels, that were associated with
aPL levels, while protein C, AT III and plasminogen were in
normal range in all patients [190].
The reason for acquired free protein S deficiency in these
patients was not clear. The proposed mechanisms included
decreased production, increased consumption, increased level
of C4b-binding protein (C4bBP), and increased systemic
clearance. As other proteins synthesized by the liver like
protein C, ATIII and plasminogen were in normal values the
reduced free protein S levels were suggested to be due to
increased C4bBP. Accordingly, the equilibrium between free
and bound protein S was shifted toward the bound form,
which is unable to function as a cofactor for activated protein
C [190].
Induced protein S deficiency on the basis of excessive
uptake by C4bBP has been reported in sepsis, DIC, SLE,
pregnancy, or during the use of oral contraceptives [194].
Antibodies which bind directly to protein S are detected and
implicated in protein S inhibition independently of other
antiphospholipid antibodies [4].
The thrombotic events in APS patients are often
associated with acquired protein S deficiency [190, 191].
Acquired protein S deficiency is established also in patients
with HIV infection, and, in some of them associated with
high aPL antibodies [192, 193].
In conclusion, these data suggest an implication for
protein S deficiency in APS thrombotic events. The
autoantibodies (anti- β2GPI) may be responsible for
decreased protein S activity particularly in conditions,
associated with increased levels of C4bBP and clinical
manifestations of the syndrome. The recent advances in
coagulation dysregulation in sepsis, focusing on the role of
protein C pathway are discussed by Diehl et al. [202].
As the physiological function of protein C is potentiated
by its cofactor protein S, the clinical data for acquired
protein S deficiency are substantial [194, 195].
There is a suggestion that protein S deficiency may be
due to dysregulation of binding of the protein with its
regulatory protein (C4bBP), one of the modulators of the
complement system. Atsumi et al. [191] showed that human
monoclonal aCL antibodies block the effect of β2GPI on the
protein S/ C4bBP system. β2GPI significantly
downredulated the binding between protein S/ C4bBP, and
5.2.2. Impaired Fibrinolysis
Impaired fibrinolysis is supposed to play an important
role in the pathogenesis of thrombosis in APS [171].
Fibrinolysis is contingent on the availability of free tissue
plasminogen activator (tPA) and of active plasminogen
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Figure (2). Stepwise schematic representation of the procoagulant role of aPL antibodies in protein C/S anticoagulant pathway.
A. Normally activated protein C (APC) forms a complex with protein S (PS), that has an anticoagulant activity, inactivating
coagulation factors Va and VIIIa. B. C4b – binding protein (C4bBP) binds to PS into an active complex, leading to PS deficiency. C.
β2GPI reduces the binding of PS to C4bBP, exhibiting an anticoagulant effect. D. APL antibodies bind to β2GPI, thus abolishing its
anticoagulant role on the protein C/S anticoagulant pathway.
activator inhibitor (PAI), its main circulating inhibitor
[186]. Macko et al. in a stroke group of patients with a
preceding infection observed reduced tPA activity, and a
lower ratio of tPA/PAI-1, indicating reduced fibrinolytic
activity. Some studies found increased release of PAI-1
without affecting the release of tPA after endothelial cell
activation in patients with connective tissue disease,
including APS [203]. Others revealed impaired release of
tPA and enhanced release of PAI-1, suggesting imbalance of
tPA/PAI-1 ratio in APS [204]. Monoclonal aCLs inhibited
fibrinolytic activity by elevating PAI-1 activity and
inhibited APC anticoagulant activity as well [205].
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Using a chromogenic assay for measurement of extrinsic
fibrinolysis Ieko et al. [206] demonstrated that monoclonal
aCL decreased the activity of extrinsic fibrinolysis in the
presence of tPA, plasminogen, fibrin and β2GPI. Addition
of monoclonal aCL in the presence of β2GPI showed
significantly lower activity of intrinsic fibrinolysis in
euglobulin fractions from APS patients [207]. These data
suggest that impairment of intrinsic and extrinsic
fibrinolysis induced by pathogenic anti- β2GPI antibodies is
one of the mechanisms for thrombosis in APS patients
[171]. On cultured EC monolayers Patterson et al. [208]
examined the effect of antiphospholipid antibodies on fibrin
formation and lysis, and determined PAI-1 in supernatants.
Four of the 14 aPL antibody-positive sera induced
prolongation of fibrin clot lysis time, and secretion of PAI-1
significantly correlated with clot lysis time on EC. The
authors suggested that impaired endothelial fibrinolysis is a
potential prothrombogenic mechanism in subjects with aPL
antibodies.
antibodies induced significant adhesion of leucocytes to EC.
Mice, deficient in complement components C3 and C5 were
resistant to enhanced thrombosis and EC activation induced
by aPL antibodies [215,216].
5.2.4. Oxidative Stress
Former assumptions that most aPL antibodies are
directed against negatively charged phospholipids recently
has been reconsidered [2, 217]. New targets of aPL
antibodies were identified as lyso(bis)phosphatidic acid
[218], sulfatides, acidic glycosphingolipids [219]. The
contemporary concepts concerning some new mechanisms of
induction of aPL antibodies and their antigenic targets are
comprehensively reviewed by Valesini et al. [2].
Another proposed mechanism through which aPL
antibodies promote thrombosis focuses on oxidant-mediated
injury. According to Horrko et al. [210] anticardiolipin
antibodies bind to oxidized, but not native cardiolipin,
suggesting that aCL antibodies recognize oxidized
phospholipids and phospholipids-binding proteins.
5.2.3. Complement Pathway
In 1997 Hasunuma et al. [221] first reported that β2GPI
bound directly to oxidized LDL (oxLDL), and that the
complex oxLDL-β2GPI was subsequently recognized by
anti-β2GPI antibodies and taken by macrophages. Later the
authors revealed that ω-carboxyl variants of 7-
ketocholesteryl esters are ligands for β2GPI and mediate the
antibody-dependent uptake of oxLDL by macrophages [222].
It is established that activated complement components
have the capacity to bind and activate endothelial and
inflammatory cells and induce a prothrombogenic state,
either directly through the membrane attack complex
(MAC), or through C5a receptor (CD88) – mediated effects
[209, 210].
In normal pregnancy, the human placenta is subjected to
complement – mediated immune attack and appropriate
complement inhibition is necessary [211]. This is supported
by the finding, that the deficiency of Crry (complement-
receptor 1-related gene/protein y, a membrane-bound
intrinsic complement regulatory protein that blocks C3 and
C4 activation results in embryonic death in pregnant mice
[212].
Some anticardiolipin antibodies cross-react with oxidized
LDL, and autoantibodies to oxidized LDL occur in
association with aCL antibodies [223]. Affinity purified
cardiolipin-binding antibodies show heterogeneity in
binding to oxididized LDL [224].
A study evaluating the antioxidant susceptibility of
subjects with aPL treated with probucol (a cholesterol
lowering agent bearing antioxidant properties) showed that
F2-isoprostanes, a marker of lipid peroxidation, significantly
decreased after treatment. These data support the role of
oxidative sensitive mechanisms in the pathogenesis aPL
induced vasculopathy and the potential effects of antioxidant
treatment [225].
Experimental studies proved the involvement of
complement in the thrombogenic effects of aPL antibodies.
Using a murine pregnancy model of BALB/c mice Holers et
al. [213] found that treatment with human IgG aPL
antibodies caused a nearly fourfold increase in the frequency
of fetal resorption, while simultaneous treatment with Crry-
Ig prevented aPL antibody-induced pregnancy losses. The
authors concluded that complement blockade at the point of
C3 activation prevents fetal loss and growth retardation in
pregnant mice with APS model, induced by passive transfer
of human aPL antibodies. Based on these findings the
authors proposed several mechanisms for the pathogenic
effects of aPLs on pregnancy outcome: 1. aPL antibodies are
preferentially targeted to the placenta; 2. placental aPLs
promote platelet and endothelial activation and directly
induce procoagulant activity through elements of the
coagulation pathway and 3. activation of the complement
pathway by aPL- Igs amplifies these effects by stimulating
the generation of further mediators from platelets and EC,
including C3a, C5a and C5b-9 MAC. C5a and C5b-9 MAC
themselves have effects on platelets and EC [214].
The major function of paraoxonase (PON), an enzyme,
carried in plasma by HDL is to prevent oxidation of LDL
[226]. In non-lupus murine model was found that PON
activity and NO (sum of nitrate and nitrite) levels were
reduced in the groups, treated with human aCL. In primary
APS, PON activity was decreased and correlated inversely
with aCL titres and directly with the total antioxidant
capacity of plasma [227] This was the first study that
focused on the intimate relationships between
oxidative/nitrosative pathways and aPL and could be
explored to test the association with vascular manifestations
of APS [228].
Some of the suggested mechanisms of thrombosis in
APS are summarized in Table 2.
CONCLUSION
Recently was shown that complement activation
mediates two important effects of aPL antibodies: induction
of thrombosis and endothelial activation. This was
demonstrated on an in vivo model of thrombosis and a
mouse model of endothelial cell activation, in which aPL
Both clinical and experimental data showed that the
procoagulant status in patients with APS is a consequence of
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Prothrombogenic Mechanisms of Antiphospholipid Antibodies Current Medicinal Chemistry, 2007, Vol. 14, No. 7 823
Table 2. Suggested Mechanisms of Thrombosis in APS
Molecular mechanisms
Activation of endothelial cells (EC)
Enhanced expression of adhesion molecules by EC and adherence of monocytes and neutrophils to vascular endothelium
Enhanced monocyte activation and adherence
Enhances platelet activation with consequent aggregation
Humoral mechanisms
Enhanced tissue factor expression
Inhibition of APC activation
Inhibiton of the protein C/S anticoagulant pathway
Inhibition of fibrinolysis
Inhibition of protein Z anticoagulant pathway
Inhibition of β2GPI anticoagulant activity
Enhanced expression of vWF
Activation of complement
Oxidant-mediated injury
Secretion of cytokines
Altered prostacycline/thromboxane balance
different pathogenetic mechanisms and pathways. Each of
the three components of the hemostatic balance- the
coagulation cascade, the anticoagulant system and
fibrinolysis are affected in subjects with circulating aPL
antibodies. The pathogenicity (thrombogenicity) of aPL
antibodies evidently depends on various factors as: 1) the
great heterogeneity of the aPL antibodies now commonly
accepted; 2) the antibody isotype and titre; 3) the specificity
of aPL antibodies to different phospholipids and
phospholipid-binding proteins, with β2GPI being the major
target antigen; 4) different target antigens evidently interfere
with various procoagulant/anticoagulant pathways involving
both cellular and humoral factors; 5) additional factors as
infections, increased apoptosis or oxidative stress may
trigger thrombosis in the presence of circulating aPL
antibodies. Moreover, the difficulty to detect patients at risk
is associated with the fact, that the routinely determined
autoantibodies are LAC, aCL and anti-β2GPI antibodies,
which significantly restricts the spectrum of the actual
circulating aPL antibodies, found in APS patients. In this
respect the efforts should be directed to the introduction of
additional laboratory tests in routine laboratory practice and
new therapeutic approaches interfering with intracellular
events of activation.
EC = Endothelial cells
HUVEC = Human umbilical vein endothelial cell
ICAM = Intercellular adhesion molecule
LAC = Lupus anticoagulant
MI = Myocardial infarction
PAI = Plasminogen activator inhibitor
PE = Pulmonary embolism
PI = Phosphatidylinositol
PS = Phosphatidylserine
SCR = Short consensus repeat
SLE = Systemic lupus erythematosus
TF = Tissue factor
TM = Thrombomodulin
tPA = Tissue-type plasminogen activator
VCAM-1 = Vascular cell adhesion molecule-1
vWF = von Willebrand factor
β2GPI = beta2 Glycoprotein I
ABBREVIATIONS
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Received: September 01, 2006Revised: November 01, 2006 Accepted: November 01, 2006
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