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Thrombotic Microangiopathy:
A Focus on Atypical Hemolytic Uremic Syndrome
Bradley P. Dixon, MD
Division of Nephrology and Hypertension, Cincinnati Children’s Hospital Medical Center
Case Presentation
• 11 year old white male, previously healthy,
presents to the emergency department with
3d of vomiting and abdominal pain, and
developed fever and dark urine today. No
diarrhea.
• ROS significant for headache, sore throat,
bleeding gums
• PMHx and PSHx signficant for febrile seizures
1 year ago, dental extractions five days ago
• Family history unremarkable.
Case Presentation, continued
• Physical exam demonstrates BP 109/72 HR 87,
with soft, nontender abdomen. No pedal
edema noted. No scleral icterus or jaundice.
• Initial labs:
– Platelet count of 18K, H/H of 12.8/36.5, nl WBC
– LDH 2445 U/L, 1+ schistocytes. Normal CPK.
– Mild/moderate renal dysfunction with SCr 1.42
– Large blood and >300 protein on urinalysis
• Diagnosis = TMA
• Differential Dx? Additional workup?
Thrombotic Microangiopathy (TMA)
• Convergence of many different
pathomechanisms
– Common link is endothelial injury
– Platelet activation and aggregation
in microvasculature
– Fibrin deposition
– Mechanical trauma to RBC
– Occurs in many microvascular beds,
but renal microvasculature
especially susceptible to this process
Thrombotic microangiopathy (TMA):
Differential Diagnosis
STEC-HUS
Shigatoxin (E coli
H0157:H7; H0104:H4)
“Primary” or
aHUS
Acquired TTP
(antibody induced
ADAMTS13 deficiency)
Infections
S pneumoniae,
HIV, H1N1 influenza A
Congenital TTP
(ADAMTS13 deficiency)
Stem Cell
Transplant
TMA
Others
HELLP, Methyl
malonic aciduria,
antiphospholipid
antibody
syndrome
•
•
•
•
Vasculitis
(SLE & other Collagen vascular
diseases)
Drugs
(calcineurin inhibitors,
ticlopidine, clopidogrel)
DIC with multiorgan failure
Thrombocytopenia
Microangiopathic hemolytic anemia
Thrombi in the microvasculature
Organ dysfunction
Modified from Besbas et al. Kidney International 2006;70:423-431.
The History of TTP & HUS
• 1925 - Moschcowitz described a new fatal disorder in a 16 year-old
girl associated with acute fever, severe anemia, heart failure and
stroke. Pathology showed thrombosis of the terminal arterioles and
capillaries of multiple organs.
• 1947 – Singer emphasized the role of platelets and coined the term
“thrombotic thrombocytopenic purpura (TTP)”.
• 1955 - Gasser et al described the hemolytic uremic syndrome (HUS),
noting platelet-fibrin thrombi in microvasculature.
• 1981 – Hypocomplementemia and Factor H deficiency identified in a
case of HUS without diarrheal prodrome.
• 1982 – Moake described the association of chronic relapsing TTP with
ultra-large VWF multimers (ULVWF) and hypothesized a defect in
vWF processing leading to platelet aggregation.
• 1985 – Link between HUS and Shigatoxin producing E coli discovered.
The Problem with Names
• Diarrheal/D+ vs. Non-diarrheal/D- HUS
– Diarrheal illness can serve as trigger for atypical HUS
• HUS/TTP
– Both aHUS and TTP can occur in adults and children
– Renal dysfunction can be either severe or mild in
aHUS
– CNS manifestations may occur in either HUS or TTP
• Current preferred terms are atypical HUS, TTP,
and STEC-HUS
The Problem with Names:
Why do we care anyway?
• Morbidity and mortality are
significant in untreated patients
• Early effective therapy can
minimize long-term morbidity
and organ damage
• Therapeutic implications of diagnostic certainty
– Plasmapheresis vs. eculizumab
• Prognostic implications of diagnostic certainty
– aHUS and TTP likely to recur, whereas STEC-HUS is not
Clinically Distinguishing aHUS and TTP
aHUS
TTP
Platelet Count
Mildly decreased or normal
Severely decreased
Lung Involvement
Often seen
Almost never seen
Renal Involvement
Generally prominent
Generally mild
CNS Involvement
Usually mild
Usually prominent
Atypical HUS: Clinical Features
• Onset
– Fulminant in 80% of patients
– Indolent in 20% of patients
– Most present with clinical triad
– Microangiopathic hemolytic anemia,
thrombocytopenia, acute kidney injury
• Extrarenal manifestations are common
– CNS, cardiovascular morbidity in ~20% of patients
– Diarrhea may be present in 30% of patients
• Very high recurrence rate in kidney transplant
Atypical HUS and Complement Biology
• Linked to uncontrolled activation of the
alternative pathway of complement system
– Up to 70% of patients have an identifiable defect
in complement regulation
• Triggering event typically necessary
– Infections, medications, surgery, pregnancy
• Uncontrolled complement activation on
endothelium leads to injury, features of TMA
Primer on Complement:
Alternative Pathway
C6-C9
C3a
C3
C3b
Ba
C3a
C3a C5aC3a
C3
C3C5C5b
Factor D C3
Factor B C3b
Bb
Properdin
C3b
Endothelial Cell
C3b
C5b
D
C3b A Bb
F
MCP
Primer on Complement:
Regulation of the Alternative Pathway
Factor H
Thrombomodulin
C3b
iC3b
Endothelial Cell
It’s Complement, Except When It’s Not…
• Diacylglycerol kinase ε (DGKE)
– Intracellular enzyme expressed
in endothelial cells, podocytes,
and platelets
– Phosphorylates arachidonic
acid–containing diacylglycerol
(AADAG), reducing activation
of prothrombotic PKC
– Not an integral component of
the complement system
Lemaire M et al,
Nature Genetics 2013
It’s Complement, Except When It’s Not…
• Mutations in DGKE recently described in a small
cohort of patients with atypical HUS
– Autosomal recessive with nearly complete penetrance
– Presented < 1 year of life, and had persistent urinary
abnormalities (microscopic hematuria and proteinuria)
– Does not demonstrate recurrence after
transplantation
Lemaire M et al,
Nature Genetics 2013
Coagulation Pathway in aHUS
• Understanding of aHUS disease biology may
be evolving as a crossroads between
coagulation and complement pathways
– THBD plays role in controlling both pathways
– Mutations in DGKE confer a prothrombotic state
in the microvasculature
– Very recently, mutations in PLG found in cohort of
36 adult aHUS patients using targeted genomic
enrichment and massively parallel sequencing (Bu
et al, JASN 2014)
Diagnostic Evaluation of TMA
• Evaluation for secondary causes
(pneumococcus, HIV, SLE)
• Assessment of complement system (aHUS)
• Assessment of ADAMTS13 (TTP)
• Assessment for Shigatoxin (STEC-HUS)
Evaluation of Complement in TMA
• Functional assessments
– CH50 (Classical pathway)
– AP50 (Alternative pathway)
• Pitfalls
– Depends on systemic consumption
– Interassay variability
• Beneficial in monitoring eculizumab therapy
Evaluation of Complement in TMA
• Quantitative assessments
– Serum C3 and C4
• Can help distinguish classical from
alternative pathway activation
– Dependent on systemic consumption
– C3 variably decreased in atypical HUS
– Serum Factor H, I, B
• Factor H decreased 15-70% in pts with CFH mutation
• Factor I occasionally decreased in pts with CFI mutation
• Factor B may be decreased with alternative pathway
activation
– May normalize with TPE before sample is drawn
Evaluation of Complement in TMA
• Quantitative assessments
– Membrane cofactor protein
expression by flow cytometry on
PBMCs
• Expression typically ~50% decreased
in heterozygous pts
• May be normal in qualitative defects
– Factor H Autoantibody by ELISA
• Detected autoantibodies may not be
biologically relevant
• Can be detected in normal individuals
• May normalize with TPE before
sample is drawn
Evaluation of Complement in TMA:
Mutational Analysis
• Most definitive method for assessing
complement
– CFH, CFI, CD46, CFB, C3, THBD, DGKE,
CFHR5, CFHR1, CFHR3
• Thrombomodulin, DGKE expressed in noncirculating cells or intracellularly
• Prognostic implications
– Progression to ESRD
• Factor H > Factor I, B > ? DGKE > MCP
– Transplant recurrence
• Factor H, I, B, C3 >> MCP, DGKE
Evaluation of Complement in TMA:
Mutational Analysis
• Challenges
– Time consuming
• Results in weeks to months
– Does not inform acute management
• TAT improving with NextGen techniques
and better bioinformatics
– Expensive
• Testing is ~$6000 at University of Iowa,
CCHMC, Blood Center of Wisconsin
• Insurance may not cover testing
Evaluation of Complement in TMA:
Mutational Analysis
• Challenges
– Mutations (variants) may not be identified
• Lack of identified mutation (~30-40% of patients with
aHUS) does not exclude aHUS or indicate lack of efficacy of
eculizumab
Evaluation of Complement in TMA:
Mutational Analysis
• Challenges
– Variants may not be biologically relevant
• “Variants of undetermined clinical significance”
– Synonymous variants associated with disease in literature
– Non-synonymous variants in which predictive algorithms disagree
on pathogenicity
– Non-synonymous variants that are common in the population
(polymorphisms)
Evaluation of Complement in TMA:
Complement Activation Biomarkers
• sC5b-9 and C5a
– Markers of terminal pathway activation
– Suppressed by adequate levels of eculizumab
• Useful for monitoring therapy
• Bb
– Marker of alternative pathway activation
• C3a, iC3b, C3c and C3d
– Markers of proximal pathway activation (C3
convertases)
Thrombotic Thrombocytopenic
Purpura (TTP)
• TTP linked to presence of ultralarge multimers of
von Willebrand Factor (Moake et al, NEJM 1982)
– Multimers remain uncleaved and bound to
endothelial cells, binding to platelets and leading to
aggregation
• Defects in the vWF cleaving protease ADAMTS13
largely responsible for TTP
– Deficient in congenital TTP (Levy et al, Nature 2001)
– Inhibited by autoantibodies in acquired TTP
(Tsai
et al, NEJM 1998; Furlan et al, Blood 1998)
Diagnostic Evaluation of TTP
• Diagnosis primarily clinical
• Historical testing methods
– Agarose gel electrophoresis of vWF multimers
– Ristocetin cofactor assay
– Collagen binding assay
– Technically challenging with interassay
variability
• More recently, ADAMTS13 biology
exploited to standardize testing
methodology
Evaluation of ADAMTS13 in TMA
• Enzymatic activity of ADAMTS13
– Most common method = cleavage of fluorogenic
modified ADAMTS13 substrate (FRET-VWF73)
– Normal result > 67% activity
– TTP < 5-10% activity
– Can be mildly to moderately decreased (10-40%) in a
number of other diseases/conditions
• DIC, liver dysfunction, sepsis/severe systemic inflammation
• Pregnancy
Evaluation of Shigatoxin in TMA
• Stool culture using
sorbitol MacConkey
agar plates
– 93% of E. coli isolates
ferment sorbitol,
whereas E. coli O157:H7
does not
– Pitfalls
• Negative in up to 50% cases of STEC-HUS
• Negative with non-O157:H7 strains that produce Shigatoxin (German
outbreak in 2011 due to O104:H4)
Evaluation of Shigatoxin in TMA
• Molecular Testing for
Shigatoxin
– ELISA/Immunoassay for
Shigatoxin
– PCR for stx1/stx2 genes
• Sensitivity and specificity > 95%
for either method
Case Presentation, continued
•
•
•
•
•
•
•
ADAMTS13 normal (82%)
Stool culture, Stx testing (EIA and PCR) negative
C3 48 (), C4 23 (nl), Factor H, I, B levels normal
Factor H autoantibody negative
CD46 FACS with ~50% expression
Genetic analysis of CFH, CFI, CFB, THBD, C3 nl
Genetic analysis of MCP reveals novel c.97+1G>A
heterozygous mutation, predicted to cause disease
Diagnosis = Atypical HUS.
How Do We
Treat This
Patient?
Plasma Therapy in aHUS
• Plasma infusion, TPE
– Long considered first-line therapy
for aHUS
• First successful uses in reversing the
disease reported nearly 30 years ago
• Mechanism of action
– Provision of non-mutant
complement regulatory proteins
– In the case of TPE, also removal of
mutant factors or autoantibodies
Plasma Therapy in aHUS
• Efficacy
– Ample anectodal evidence of efficacy
– No well-controlled prospective clinical trials
showing efficacy in aHUS
– Two early prospective trials in 1988 compared
plasma therapy with supportive care alone
• No benefit in death, ESRD, proteinuria or hypertension
• Did not distinguish between STEC-HUS and aHUS
– Likely has little to no role in aHUS caused by
membrane bound or intracellular factors
(MCP/CD46, thrombomodulin, DGKE)
Plasma Therapy in aHUS
• Pros
– Widely accepted in treatment of aHUS
– Also first-line therapy for TTP (clinical overlap)
– Available at most pediatric and adult centers
• Cons
– Complications include hyperproteinemia, catheter
related central venous thrombosis and infection,
anaphylaxis to plasma
– Some patients demonstrate continued disease activity
despite plasma therapy, or relapse after discontinuation
– Technically challenging in small children
Eculizumab in aHUS
• Eculizumab is a humanized
monoclonal antibody against
complement factor C5.
– FDA approval in 2011 for
treatment of aHUS
• Mechanism of action
– Blockade of C5 conversion to C5a and C5b,
preventing membrane attack complex C5b-9
formation
– C3 convertase remains intact, therefore
opsonization of pathogens is preserved
Eculizumab in aHUS
• Efficacy
– Two prospective open-label multicenter industrysponsored trials with total of 37 patients
• Adults with plasma-dependent/responsive aHUS (20 pts)
• Adults with plasma-refractory aHUS (17 pts)
– Prospective open-label phase II trial with 19
pediatric patients
Eculizumab in aHUS
• Efficacy – Prospective Trials
– Hematologic normalization (platelets and LDH) on
eculizumab
• 76-90% of adult patients at median of 37 weeks (NEJM, 2013)
• 82% of pediatric patients by 26 weeks (Kidney Int, 2016)
– Renal improvement on eculizumab
• Mean eGFR improvement
– 5mL/min/1.73m2 in plasma-dependent adult patients
– 33mL/min/1.73m2 in plasma-refractory adult patients
– 64mL/min/1.73m2 in pediatric patients
• 4/5 adult patients and 9/11 pediatric patients on dialysis able
to discontinue
Eculizumab in aHUS
• Pros
– Very well tolerated (peripheral IV, infusion length 35 min,
no premedication necessary)
– Highly effective, even in patients on dialysis from aHUS
– Highly effective in aHUS patients both with and without
identified complement defect(s)
• Cons
–
–
–
–
Risk of meningococcal disease
Expensive! ($409,500/year according to Forbes)
Debate ongoing as to the optimal length of therapy
Discontinuation may lead to relapse of the disease
Liver Transplantation in aHUS
• May reconstitute complement defect in patients
with secreted complement regulators
– Factor H, Factor I, Factor B, C3
– Likely ineffectual in MCP, Thrombomodulin, DGKE
• Early reports with high perioperative morbidity and
mortality (Remuzzi et al., Lancet 2002 and others)
• Later experiences have shown more promise (Saland
et al., Am J Trans 2006; Saland et al., CJASN 2009)
– Intensive perioperative therapy with TPE, anticoagulation
What’s on the Horizon for aHUS?
• Factor H concentrate and recombinant Factor H
• Mini-Factor H
• TT30
– Soluble recombinant fusion protein consisting of iC3bbinding region of CR2 and inhibitory domain of Factor H
•
•
•
•
Mirococept (Membrane targeting sCR1)
Recombinant soluble thrombomodulin
RA101495 (Peptide C5 inhibitor)
ACH-4471 (Small molecule Factor D inhibitor)
Who’s On First?
• What a hematologist brings to the table
– Greater experience with TTP
– Greater depth of pathomechanisms/DiffDx of
thrombocytopenia, MAHA
– May have better understanding of diagnostic
evaluation of TMA
• What a nephrologist brings to the table
– Greater experience with STEC-HUS
– Greater depth of pathomechanisms/DiffDx of renal dysfunction
– May have better understanding of complement biology
• Team-based approach at CCHMC
– Hematology, Nephrology, Critical Care
– Cooperative evaluation and development of treatment plans
Summary of Diagnostic Evaluation of TMA
• aHUS
– Quantitation of serum
complement proteins*
• C3, C4
• Factor H, Factor I, Factor B
– MCP FACS on PBMCs
– Factor H AutoAb ELISA*
– Mutational analysis
• STEC-HUS
– Stool culture
– Stx ELISA or PCR
• TTP
– ADAMTS13 activity +/inhibitor assay &
inhibitor Ab ELISA*
• Factor H, Factor I, MCP, C3,
Factor B, Thrombomodulin,
DGKE, CFHR5, CFHR1-CFHR3
deletion
*IMPORTANT! These tests should be obtained prior to the initiation
of plasmapheresis!
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