Atypical Hemolytic-Uremic Syndrome The Interplay Between Complements and the Coagulation System

Abstract

Hemolytic-uremic syndrome (HUS) is a rare life-threatening disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, and impaired renal function. A thrombotic microangiopathy underlies the clinical features of HUS. In the majority of cases, HUS follows an infection with toxin-producing bacteria such as verotoxin-producing Escherichia coli. In some cases, HUS is not preceded by a clinically apparent infection, and therefore, is named atypical HUS. The prognosis of atypical HUS is poor. While mortality approaches 25% during the acute phase, end-stage renal disease develops in nearly half of patients within a year. Evidence is accumulating that complement activation through the alternative pathway is at the heart of the pathophysiology leading to atypical HUS. Genetic abnormalities involving complement regulatory proteins and complement components form the molecular basis for complement activation. Since microvascular thrombosis is a quintessential feature of atypical HUS, complements and the coagulation system must work in tandem to give rise to the pathologic alterations observed in this condition. Here, a brief discussion of clinical and morphologic features of atypical HUS is followed by a concise presentation of the complement and coagulation systems. The interplay between complements and the coagulation system is graphically highlighted. Last but not least, conventional and emerging therapies for atypical HUS are outlined.

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KIDNEY DISEASES
340
Iranian Journal of Kidney Diseases | Volume 7 | Number 5 | September 2013
Review
Atypical Hemolytic-Uremic Syndrome
The Interplay Between Complements and the Coagulation System
Ali Nayer,
1
Arif Asif
2
Hemolytic-uremic syndrome (HUS) is a rare life-threatening
disorder characterized by microangiopathic hemolytic anemia,
thrombocytopenia, and impaired renal function. A thrombotic
microangiopathy underlies the clinical features of HUS. In the
majority of cases, HUS follows an infection with toxin-producing
bacteria such as verotoxin-producing Escherichia coli. In some
cases, HUS is not preceded by a clinically apparent infection, and
therefore, is named atypical HUS. The prognosis of atypical HUS is
poor. While mortality approaches 25% during the acute phase, end-
stage renal disease develops in nearly half of patients within a year.
Evidence is accumulating that complement activation through the
alternative pathway is at the heart of the pathophysiology leading
to atypical HUS. Genetic abnormalities involving complement
regulatory proteins and complement components form the molecular
basis for complement activation. Since microvascular thrombosis
is a quintessential feature of atypical HUS, complements and the
coagulation system must work in tandem to give rise to the pathologic
alterations observed in this condition. Here, a brief discussion of
clinical and morphologic features of atypical HUS is followed by a
concise presentation of the complement and coagulation systems.
The interplay between complements and the coagulation system
is graphically highlighted. Last but not least, conventional and
emerging therapies for atypical HUS are outlined.
IJKD 2013;7:340-5
www.ijkd.org
1
Division of Nephrology,
University of Miami, Miami, FL,
USA
2
Division of Nephrology, Albany
Medical College, Albany, NY,
USA
Keywords. atypical hemolytic-
uremic syndrome, complement
system proteins, blood
coagulation disorders
INTRODUCTION
Thrombotic microangiopathy occurs in several
distinct clinical settings such as hemolytic-uremic
syndrome (HUS), thrombotic thrombocytopenic
purpura, antiphospholipid syndrome, scleroderma
renal crisis, and malignant hypertension.
Hemolytic-uremic syndrome is characterized by
endothelial injury and microvascular thrombosis
resulting in microangiopathic hemolytic anemia,
thrombocytopenia, and ischemic injury to organs,
especially to the kidneys.
1,2
In the majority of
patients with HUS, a bacterial infection such as
gastroenteritis, often hemorrhagic, due to Shiga
toxin-producing Escherichia coli precedes thrombotic
microangiopathy within a week. In approximately
10% of patients with HUS, a preceding bacterial
infection is not identified (known as atypical HUS).
1,2
Accumulating evidence indicates an important role
for the complement system in the pathogenesis
of atypical HUS.
1,2
The reciprocal interactions
between complements and the coagulation system
might provide the molecular basis for vascular
thrombosis observed in atypical HUS.
CLINICAL PRESENTATION
Clinical manifestations of atypical HUS are a
consequence of microvascular thrombosis resulting
in ischemic injury and microangiopathic hemolysis
(Figure 1).
2
Atypical HUS can occur at any age.
While usually abrupt in onset, the presentation can
be insidious in nearly 20% of patients. A hemoglobin
concentration below 10 g/dL, a platelet count below

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Iranian Journal of Kidney Diseases | Volume 7 | Number 5 | September 2013
150 × 10
9
/L (usually between 30 × 10
9
/L and 60
× 10
9
/L), and impaired kidney function are often
found on presentation. Laboratory tests also disclose
features of intravascular hemolysis including
elevated serum lactate dehydrogenase and reduced
serum haptoglobin levels. Fragmented erythrocytes
(schistocytes) and reticulocytes are seen on blood
films. Serum concentration of complement C3 may be
reduced. The most frequent manifestations of kidney
disease in atypical HUS are azotemia, hypertension,
proteinuria, and hematuria. Kidney function is
frequently severely impaired necessitating renal
replacement therapy. Hypertension could be
severe as a result of hyperreninemia and volume
expansion. Proteinuria can be in the nephrotic
range. Extrarenal manifestations occur in about
20% of patients. Neurological manifestations occur
in approximately 10% of patients and include
altered mental status from drowsiness to come,
focal neurological deficits, and seizure. Cardiac and
distal limb ischemia can occur in some patients. A
catastrophic presentation due to the involvement
of multiple organs is observed in approximately
5% of patients.
PATHOLOGICAL FEATURES
Histologically, atypical HUS is indistinguishable
from HUS caused by toxin-producing bacteria.
Although atypical HUS can affect various vascular
beds, pathologic features in the kidney have been
Figure 1. Renal thrombotic microangiopathy in atypical hemolytic-uremic syndrome (Courtesy of Xu Zeng, MD, PhD, Nephrocor
Bostwick Laboratory). A and B, microthrombi (arrows) in glomerular capillaries. C, immunofluorescence micrograph revealing a fibrin
thrombus (arrow) in a glomerular capillary. D and E, thrombosis (arrows) of 2 small arteries. F, immunofluorescence micrography
revealing a fibrin thrombus (arrow) partially occluding a small artery. G, electron micrography demonstrating a thrombosed (white
asterisk) glomerular capillary loop. A patent glomerular capillary (black asterisk) contains 3 platelets attached to each other (black
arrows). A podocyte (white arrow) is seen in the urinary space. (A and D, hematoxylin-eosin; B and E, Masson’s trichrome; and C and F,
antiserum against fibrin/fibrinogen).

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best studied.
3
In the acute phase, there is thickening
of glomerular capillary walls and arterioles due to
accumulation of plasma proteins including fibrin
or fibrinogen in the subendothelial zone. This is
in part due to the loss of structural and functional
integrity of vascular endothelial cells. Thrombi can
be identified in glomerular capillaries, arterioles,
or arteries (Figure 1). However, thrombi are not
necessary for making a histologic diagnosis of atypical
HUS. Endothelial cell swelling or denudation often
accompanies thrombosis. Glomerular capillaries and
arterioles can undergo fibrinoid necrosis consisting
of fibrin, cellular debris, and rare neutrophils.
Arcuate and interlobular arteries frequently
develop edematous or mucoid intimal expansion
resulting in the narrowing of vascular lumen.
Immunohistological examination demonstrates
irregular deposition of fibrin, immunoglobulin
M, C3, and C1q in areas of fibrinoid necrosis and
edematous intimal expansion. In the subacute
phase, mesangial cell interposition and formation
of new basement membrane material results in
remodeling of glomerular capillary walls. These
changes are reminiscent of glomerular structural
alterations observed in membranoproliferative
glomerulonephritis, although with less glomerular
hypercellularity. Chronic glomerular injury leads to
segmental or global glomerular sclerosis. Tubular
atrophy and interstitial fibrosis prevail. Collagen
deposition in the intima of arterioles and arteries
give rise to arteriolosclerosis and arteriosclerosis,
respectively. Concentric laminations in the fibrotic
intima often result in an “onion skin” pattern of
vascular injury.
THE COMPLEMENT SYSTEM
Overview
An evolutionary conserved part of the immune
system, complements represent a first-line defense
system against invading pathogens.
4
Initially
recognized for their complementary bactericidal
activity, complements are positioned in the heart
of an intricate network of biological systems that
regulate innate and adaptive immunity, waste
disposal, angiogenesis, regenerative processes, and
lipid metabolism. Complements are activated through
the classical, lectin and alternative pathways (Figure
2).
4
The classical pathway is strongly activated by
immune complexes, which are recognized by
the versatile pattern recognition molecule C1q.
Carbohydrates such as those present on microbial
surfaces activate the lectin pathway. Following target
recognition, proteolytic cleavage of C4 and C2 results
in generation of the classical and lectin pathway
C3 convertase (C4b2b).
4
The alternative pathway is
activated by complex polysaccharides such as those
present on the surface of microorganisms. Factor
B, factor D, and C3 participate in generation of the
alternative pathway C3 convertase (C3bBb), which
is stabilized by factor P (also known as properdin).
Complement C3 cleavage by the C3 convertases
and subsequent C5 cleavage by the C5 convertases
results in the formation of C5a and C5b. The latter
participates in the assembly of the membrane attack
complex (membrane attack complex [MAC], C5b-9,
and terminal complement complex). Regardless of
origin, all surface-bound C3 convertases can induce
activation of the alternative pathway. Therefore,
the alternative pathway plays a dominant role in
the total complement activity.
Physiologic activities of the complement
system include host defense against infections,
waste disposal, and connection between innate
and adaptive immunity.
4
In the context of an
immune response, anaphylatoxins C3a and C5a
trigger proinflammatory signaling and attract
neutrophils, monocytes, and macrophages to
the site of complement activation. Opsonin C3b
facilitates phagocytosis, while MAC mediates
target cell activation, injury, or lysis in a dose-
dependent manner.
Soluble and membrane-bound factors regulate
the activity of complements (Figure 2).
5
Soluble
complement regulatory proteins include factor I (FI),
factor H (FH), and C4-binding protein. Synthetized
mainly in the liver, FI is a serine protease that
suppresses complement activity by breaking down
fluid-phase and cell-bound C3b and C4b. Cofactors
are required for the catalytic activity of factor I. A
155-kDa glycoprotein synthesized mainly in the
liver, FH serves as a cofactor for FI and facilitates
FI-mediated C3b degradation. By removing Bb from
C3bBb, FH also accelerates decay of the alternative
pathway C3 convertase. Complement C4-binding
protein has similar effects on the classical and lectin
pathway C3 convertase. Complement regulatory
proteins are also found on the surface of most
human cells. Membrane cofactor protein (CD46) is a
membrane protein expressed by all cells except for
erythrocytes. Membrane cofactor protein binds C3b

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and C4b and serves as a cofactor for FI. Complement
receptor 1 (CR1, CD35, C3b/C4b-receptor) serves as
a cofactor for FI and accelerates decay of convertases.
Thrombomodulin, a membrane glycoprotein with
anticoagulant activity, also facilitates FI-mediated
C3b inactivation.
Complement Abnormalities in Atypical
Hemolytic-Uremic Syndrome
Reduced C3 and normal C4 levels in the serum
of some patients with atypical HUS led to the
notion that complements are activated through
the alternative pathway. Subsequent work
demonstrated that approximately half of patients
with atypical HUS have hereditary genetic defects
involving soluble and membrane-bound proteins
that regulate complement activity (Table).
1,2
Loss
of function mutations involving FH, FI, membrane
cofactor protein, and thrombomodulin have
been associated with atypical HUS. Enhanced
complement activity could also be the result
of increased activity of individual complement
components. Of note, increased FB and C3 activities
have been associated with atypical HUS. In some
patients with atypical HUS, autoantibodies directed
against FH are found. Anti-FH antibodies block the
binding of factor H to C3b resulting in unopposed
assembly of C3bBb and complement activation
through the alternative pathway.
Figure 2. Complement activation and regulation. The classical, lectin, and alternative pathways of complement activation are
demonstrated. The phases of complement activation are also shown. Regulatory proteins are in black circles and ovals. The T sign
denotes an inhibitory effect. See text for details. MBL, indicates mannose-binding lectin; MCP, membrane cofactor protein; C4BP, C4-
binding protein, FI, factor I; MAC, membrane attack complex; TM, thrombomodulin; FH, factor H; TAFI, thrombin-activatable fibrinolysis
inhibitor; FD, factor D; FB, factor B; FP, factor P; DAF, decay accelerating factor; and Ab, antibody.
Prevalence, %
Complement Abnormality Nonfamilial Familial
Factor H 15 to 20 40 to 50
Anti-factor H antibodies 6 to 10 Not known
Membrane cofactor protein 6 to 10 7 to 15
Complement C3 4 to 6 8 to 10
Factor I 3 to 6 5 to 10
Thrombomodulin 2 9
Factor B Rare 1 to 2
Complement Abnormalities in Atypical Hemolytic-Uremic
Syndrome

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Interplay Between Complements and
Coagulation System
Unleashed activation of complements in atypical
HUS culminates in intravascular coagulation.
Although an association between inflammation
and a hypercoagulable state has long been
recognized, the interplay between complements
and the coagulation system has recently come
to light
(Figure 3).
6,7
It has been shown that
complement activation promotes intravascular
coagulation. In return, coagulation reactions can
stimulate complement activation. A potent trigger
of inflammation, anaphylatoxin C5a induces
expression of tissue factor (TF) on endothelial cells,
monocytes, and neutrophils (A in Figure 3). A
membrane protein, TF is the receptor and cofactor
for coagulation factor VII and its activated form
VIIa.
8
Factor VIIa-TF complex triggers activation
of coagulation factors X and IX (B in Figure 3).
On the surface of the TF-bearing cells, activated
factor X (Xa) assembles with activated factor V
(Va) to form the prothrombinase complex (Va-Xa).
This activates coagulation factor II (prothrombin)
to generate a small amount of thrombin (IIa) (C
in Figure 3). A multifunctional serine protease,
thrombin plays a pivotal role in coagulation and
cellular activation.
8
A potent platelet agonist,
thrombin binds to protease-activated receptors
and induces platelet activation, adhesion, and
aggregation (D in Figure 3). In addition, thrombin
triggers exocytosis of platelet α granules containing
coagulation factors I, V, and VIII (E in Figure 3). It
also activates several coagulation factors, including
factors V, VIII, XI, XIII, and plasminogen. Assembly
of activated factor IX (IXa) with activated factor VIII
(VIIIa) on the surface of platelets causes activation
of factor X (Xa; F in Figure 3). The prothrombinase
complex (Va-Xa) formed on the surface of platelets
converts prothrombin to thrombin (G in Figure 3).
Thrombin cleaves fibrinogen to fibrin and induces
activation of coagulation factor XIII, which cross-
links and stabilizes fibrin (H and I in Figure 3).
Reciprocal interactions between platelets and
complements are of particular interest in the
Figure 3. Interplay between complements and the coagulation system. Complement components are labeled by Arabic numerals in
grey circles. Coagulation factors are indicated in yellow rectangles except for thrombin, which is in a purple rectangle. A lower case a
following Roman numerals stands for activated. An inhibitory effect is indicated by an inverted T sign. A red star indicates an activating
effect. See text for details. PAI-1 indicates plasminogen activator inhibitor-1; TF, tissue factor; TPA, tissue plasminogen activator; UPA,
urokinase plasminogen activator; FDP, fibrin degradation products; PLG, plasminogen; PN, plasmin; PS, P-selectin; MP, microparticle;
VWF, Von Willebrand factor; α, platelet alpha granule; δ, platelet delta granule; and PAR, protease-activated receptor.

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pathogenesis of atypical HUS.
6,7
Platelets can
cleave C3 into its active components C3a and
C3b (J in Figure 3). Binding of C3a to its receptor
on the surface of platelets stimulates activation,
adhesion, and aggregation of platelets (K in Figure
3). C5b participates in the assembly of MAC on
the surface of platelets resulting in generation of
negatively charged prothrombotic phospholipids
in the cell membrane (L in Figure 3). Membrane
attack complex also triggers secretion of platelet
storage granules and release of the TF-bearing
microparticles (L in Figure 3). Binding of C1q to
its receptor on the surface of platelets stimulates
expression of P-selectin and integrins such as
GPIIb-IIIa (M in Figure 3). A docking site for C3b,
P-selectin can facilitate formation of C5 convertase
of the alternative pathway of complement activation
(N in Figure 3). Platelets also stabilize C3b through
phosphorylation (O in Figure 3). Complement C5a
stimulates production of plasminogen activator
inhibitor 1 in mast cells and basophils (P in Figure 3).
A potent inhibitor of tissue plasminogen activator
and urokinase plasminogen activator, plasminogen
activator inhibitor 1 suppresses generation of
plasmin and breakdown of fibrin (Q in Figure 3).
PROGNOSIS AND TREATMENT
The prognosis of atypical HUS is poor. While
mortality approaches 25% during the acute phase,
end-stage renal disease develops in nearly half of
patients within a year.
1,2
The outcome of kidney
transplantation in patients with atypical HUS is
disappointing.
9
Depending on the underlying genetic
abnormality, the recurrence rate in renal allograft
could be as high as 80%. In the vast majority of
cases, recurrent atypical HUS leads to graft loss.
Plasma therapy including plasma exchange and
infusion has remained the standard treatment for
atypical HUS.
1,2
While plasma infusion replenishes
deficient regulatory proteins, plasma exchange
therapy has the additional benefit of removing
factors that inhibit regulatory proteins. However,
in some cases prolonged treatment may be needed
for the induction of remission. In addition, plasma
therapy may even fail to induce remission.
Considering the pivotal role of complements in
the pathogenesis of atypical HUS, strategies to
directly suppress complement activity represent
a logical therapeutic approach. A neutralizing
monoclonal antibody directed against complement
C5, eculizumab has been shown to exert salutary
effects in patients with atypical HUS.
1,2
Eculizumab
blocks cleavage of C5 into C5a and C5b and prevents
formation of MAC resulting in attenuation of
complement-mediated inflammatory reactions and
tissue injury. It is conceivable that beneficial effects
of eculizumab in atypical HUS may also be related to
reduced activity of the coagulation system. That is,
reduced expression of tissue factor on endothelium
and immune cells, suppression of the activity of
platelets, immune cells, and endothelium, as well
as enhanced fibrinolytic activity.
CONFLICT OF INTEREST
None declared.
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Correspondence to:
Ali Nayer, MD
Division of Nephrology and Hypertension, University of Miami,
Clinical Research Building, Suite 825, 1120 NW 14th St, Miami,
FL 33136, USA
Tel: +1 305 243 8491
Fax: +1 305 243 3506
E-mail: anayer@med.miami.edu
Received May 2013
Accepted June 2013