Sacha Zeerleder, MD PhD

Internist-hematologistAcademic Medical Centre, Amsterdam Sanquin Research,

Amsterdam

Sacha Zeerleder, MD PhD

Internist-hematologistAcademic Medical Centre, Amsterdam Sanquin Research,

Amsterdam

Plasma-containing blood products in Complement-mediated diseases

beneficial or harmful?

MAC (C5b-9)

C5b

C3b

C3

Opsonization(C3b, C4b)

Inflammationanaphylatoxins

(C3a, C5a)

Complement system

Alternative pathway

Classical pathwayLectin pathway

C4bC2aC4bC2a

Low-grade hydrolysis

MAC (C5b-9)

C5b

C3b

C3

H2OBb

C3b B

C3b

P

D

P

Classical pathway Lectin pathway

Bb

C3b B

C3b

B

DAA

CA

FI

Complement system

Decay accelerating activity (DAA) for C3 convertase (C3bB/C3bC3b)• Factor H (FH)• DAF (CD55) Cofactor activity for Factor I (FI): inactivates C3b• Factor H (FH)• Membrane cofactor protein

(MCP)

Wouters & Zeerleder, Haematologica 2015

MAC (C5b-9)

C5b

C3b

C3Classical pathway Lectin pathway

Bb

C3b B

C3b

B

DAA

CA

FI

Complement system

Wouters & Zeerleder, Haematologica 2015

FI

C3bi

Non-activating

C3bFH

activating

C3b

FH

B [2.2uM]

[3.2uM]

EqualaffinityforFHandB

C3bB

Fearonetal..1977;Lawetal.1977;Volanakis1998

Complement system- amplification loop

Amplification loop- regulation

Three complement activation pathways: regulation

C1-inhibitor

C4BP

Factor I

Factor H

Plasma inhibitors

MCP (CD46)

sCR1

DAF (CD55)

protectin (CD59)

Membraneinhibitors

Wouters & Zeerleder, Haematologica 2015

MAC(C5b-9)

C5b

C3b

C3

Opsonization(C3b,C4b)

Inflammationanaphylatoxins

(C3a,C5a)

Alternativepathway

ClassicalpathwayLectinpathway

C4bC2aC4bC2a

MAC(C5b-9)

C5b

C3b

C3

Opsonization(C3b,C4b)

Inflammationanaphylatoxins

(C3a,C5a)

Alternativepathway

ClassicalpathwayLectinpathway

C4bC2aC4bC2a

Complement- mediated diseases and hemolysis

Wouters & Zeerleder, Haematologica 2015

Autoimmune Hemolytic Anemia (AIHA)

Cold Agglutinin Disease

Paroxysmal Nocturnal Hemoglobinuria (PNH)

Atypical Hemolytic Syndrome (aHUS)

Dysregulation alternative pathway – loss of control

PNH

PNH: deficiency of membrane-bound regulators

Wouters & Zeerleder, Haematologica 2015; Zeerleder, van Solingen & v. Wijk 2015

MAC(C5b-9)

C5b

C3b

C3

Opsonization(C3b,C4b)

Inflammationanaphylatoxins

(C3a,C5a)

Alternativepathway

ClassicalpathwayLectinpathway

C4bC2aC4bC2a

MCP (CD46)

DAF (CD55)

protectin (CD59)

Membraneinhibitors

CD alternative name function

CD 14Pattern recognition

receptor (PRR)Receptor for

LPS

CD 16 Fc-gamma receptor IIIbLow-affinity receptor IgG

CD 24 Heat stable antigen Cell adhesion

molecule

CD 48 Signaling lymphocyte activation molecule 2

(SLAMF2)

Member Ig-superfamily

CD 52 Campath-1Not entirely

clear

CD 55Inhibition formation C3-

convertaseCell-adhesion

CD 59Membrane inhibitor of

reactive lysis

Inhibition C9 polymerization

(MAC)

CD 66 CEA family Cell-adhesion

Etn

Man

Man

ManGluc

In

GPI-linked protein

PIGA -gen

Membrane bound regulators are GPI- linked

Zeerleder, van Solingen & v. Wijk 2015

C3b MACC5b

PNH patients with hemolytic disease (n=29)

• NO consumption (12-fold)

• PAP 41% (Echo)

• RV function 80% (MRI)

• 60% PE

The dark side of complement activation and cell-free heme

Thromboembolism

• Accounts for 40-67% of deaths

• 29-44%: ≥ 1 TEE in the course of disease

• TEE at presentation: 4-yrs survival 40%

• 19% of patients: TEE is a herald of PNH

• It can occur ant any site:

• Multiple thrombosis 20%

Hill et al. 2010; Hill et al. 2012; Araten et al. 2005, de Latour et al. 2008, Hillmen et al. 1995, Moyo et al. 2004, Nishimura et al. 2004, Parker et al. 2005, Socie et al. 1996, Poulou et al. 2008, Gralnick et al. 1995, Hill et al. 2013, Zhao et al. 2013

Inhibition of complement-mediated hemolysis: anti-C5

Eculizumab

• Humanized monoclonal antibody

• Blocks cleavage of C5

- decrease in C5a

- decrease in C5b9

Hillmen et al. 2004, Hillmen et al. 2007, Rother et al. 2007, Mache et al 2009, Legendre et al. 2013

Anti-C5

MAC (C5b-9)

C5b

C3b

C3

Bb

C3b B

C3b

P

D

P

Classical pathway Lectin pathway

Exacerbation complement activation in PNH

Roth et al. Int J Hematol. 2011; Kelly et al. NEJM 2015 ; Wouters & Zeerleder, Haematologica 2015

C3b

C5b

MAC (C5b-9)

Exacerbation due to:• Infection• Pregnancy• Surgery• Trauma

Plasma-containing blood products?

5-10 mlWashed: 0 ml

Apheresis: 150-400 mlPAS III/E: ~127ml/~110ml

concentrated: <5 ml

200 ml

Amount of plasma per unit

How to deal with plasma-containing products in PNH?

Jackson et al. J Clin pathol 1992; Rosse, transfusion 1989; Brecher & Taswell; Transfusion 1989Kelly et al. N Eng J Med 2015; Richtlijn PNH NVH, 2016

75 pregnancies in 61 PNH patients• Platelet transfusions in 16 pregnancies• 2 post-partum hemorrhages complicated by

thromboembolic events (1 after plasma infusion)

Mayo Clinic (1950-1987)• 23 PNH patients (positive Ham test)

→ 556 blood products→ 431 RBC products

(94 whole blood, 208 packed cells, 80 units white-cell poor RBCs, 38 units of saline-

washed red cells, 5 units frozen RBCs and 6 units via intraoperative salvage)

1 episode of postransfusion hemolysis (O product to a AB recipient)

Brecher & Taswell; Transfusion 1989

Transfusion of plasma-containing products in PNH

1978: VSAA, treatment with ATG; no PNH1980: postpartum hemorrhage, transfusion 17 units washed RBC and 50 u platelet

concentrate (washed due to an earlier FNHTR)1990: cervical loop biopsy; transfusion of 4 RBC’s (O neg) (patient: A1 pos)

Acute hemolysis with Hb 4 g/dl; renal insufficiencyDiagnosis PNH (positive Ham- and sucrose lysis test)

Plasma donors (n=4) O neg (no irregular antibodies)

Induces lysis of A1 Test RBCs (titer 1:2)

Induces lysis of A1 patient RBCs (titer 1:512)

In-vitro

MAC (C5b-9)

C5b

C3b

C3

Bb

C3b B

C3b

P

D

P

Classical pathway

Minor incompatibility: exacerbation complement activation

Roth et al. Int J Hematol. 2011; Kelly et al. NEJM 2015 ; Wouters & Zeerleder, Haematologica 2015

C3b

C5b

MAC (C5b-9)

Anti-A1

A1

CP activation

Apheresis: 150-400 mlPAS III/E: ~127ml/~110ml

concentrated: <5 ml

5-10 mlWashed: 0 ml

200 ml

Amount of plasma per unit

No washing needed

Minor mismatchPAS III/E,

concentrated product

If possible: avoidGive additional

dose eculizumab

How to deal with plasma-containing products in PNH?

Jackson et al. J Clin pathol 1992; Rosse, transfusion 1989; Brecher & Taswell; Transfusion 1989Kelly et al. N Eng J Med 2015; Richtlijn PNH NVH, 2016

Atypical HUS

ADAMTS13Complement activation/-dysregulationEndothelilal destruction (Shiga toxine)

Thrombotic microangiopathy (TMA) - aHUS

UL WF

C5C9b

Hemolytic anemiaThrombocytopeniaFeverNeurological symptomsRenal dysfunction

MAC complex (C5b-9)

C5b

C3b

C3

H2OBb

C3b B

C3b

P

D

P

Classical pathway Lectin pathway

Opsonization(C3b, C4b)

Inflammationanaphylatoxins

(C3a, C5a)

Bb

C3b B

C3b

B

DAA

CA

FIDecay accelerating activity (DAA) for C3 convertase (C3bB/C3bC3b)• Factor H (FH)• DAF (CD55) Cofactor activity for Factor I (FI): inactivates C3b• Factor H (FH)• Membrane cofactor protein (MCP

- CD46)

Atypical HUS: complement regulation “on tilt”

Wouters D, Zeerleder S. Haematologica. 2015;100(11):1388-95

Atypical HUS : Complement Factor H (CFH)

• Complement Control Protein repeats: CCP repeats (à 60 aa)• Mw~150 kD• Chromosome 1q32• aHUS: 30% mutations in FH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20NH2 COO

Recognition-site C3b:

• dissociation C3-convertase

(C3bB, C3bC3b)

• Cofactor for CFI (cleavage C3b)

Recognition-site surfaces:

• Basement membranes

• Endothelium

20% 60%Mutations

Autoantibodies to FH

Kavanagah & Goodship Nephron Clin Pract.2010, Noris & Remuzzi, N Engl J Med 2009, Richards et al. Adv Immunol 2007

aHUS: complement regulation alternate pathway versus C5

de Jorge et al. J Am Soc Nephrol 2011

CFH -/- del16-20: Spontaneous development of aHUS

No C5 activation - no damage

Pickering et al. J Exp Med 2007

FH mutation – renal damage

No complement depositioncomplement

deposition

Genetic testing for mutations in aHUS

Penetrance ~50%

• Factor H (CAVE: occasionally auto-Ab to the C-term. FH)

• MCP/CD46 mutation

• Factor I

• C3

• FB

• Thrombomodulin

• DGKE (diacylglycerol kinase epsilon) mutation

Time consuming (but important to do)

Up to 50% no detected mutation

30%

15%

<10%

How does overt aHUS develop?

1 or more genetic mutations

Genetic polymorphisms Trigger++

Ab: Antiody; FH: Factor HKavanagah & Goodship. Nephron Clin Pract. 2011;118(1):c37-42, Noris & Remuzzi, N Engl J Med 2009;361(17):1676-8. Richards et al. Adv Immunol 2007; 96:141-77. Lemaire et al. Nat Genet. 2013;45(5):531-6.

End-stage renal failure/death in patients with aHUS

CFH

CFI

C3

No mutation

Auto-AB CFH

MCP

Marina Noris et al. CJASN 2010;5:1844-1859

aHUS mutations and response to plasma

Mutated gene/protein typeFrequency

(%)Death/ESRD

(%)Response to plasma (%)

CFH (incl. CFH/CFHR1 hybrid genes)

LCR 24-28 70-80 63%

MCP (CD46) LCR 5-9 <20 97%

CFI LCR 4-8 60-70 25%

C3 GoF 2-8 60-70 57%

CFB GoF 0-4 70

Thrombomodulin ? 0-5 50-60 88%

CHFR1/3 def. with anti-FHantibodies

LCR 13-10 30-70 75%

Diacylglycerol kinase ɛ Prothrom. 0-3 46

none 30-48 50 69%

ESRD: End stage renal disease; LCR: loss of complement regulation; GoF: Gain of function mutation (complement activation)

Noris et al. CJASN 2010; Jokiranta T. Blood 2017

Plasmapheresis: aim to restore regulators and/or remove dysfunctional protein

aHUSpatients

hemodialysis(n= 6)

Eculizumab(n= 2)

Plasmapheresis(n=3)

man 2 0 1

woman 4 2 2

Median age 52,3 25,5 16

aHUS: effects dialysis and plasmapheresis on complement activation

before after

0

500

1000

1500

nM

C3b/c

(normal <57 nM)

C4b/c

(normal < 8 nM)

C3bc C4bc

0

200

400

600

800

nM

normal <57 nM normal <8 nM

dialysis anti-C5 PF

0

50

100

150

200

nM

before

after

p<0.001

dialysis anti-C5 PF

0

200

400

600

800

nM

before

after

p<0.001

aHUS patient: transfusion of 10 units omiplasma Significant complement

activation upon procedure Complement activation

products in the product

Plasmapheresis replete deficient protein but does not prevent (low-grade) complement activation

Rethans, de Wit, Schmidt, van Merlo, Wouters, ten Brinke, Bemelmans, Zeerleder; MS in preparation

C3bc C4bc

Anti-C5 (Eculizumab) treatment in aHUSPlatelet counts

Renal function

Uncontrolled Complement Activation Has a Role in Many Diseases

1. Schrezenmeier H. Transfus Apher Sci. 2012;46:87-92; 2. Holers VM et al. Immunol Rev. 2008;223:300-316. 3. Huda R. Rev Neurosci. 2014; doi.10.15/14. Epubahead of print. 4. Meri S. European Journal of Internal Medicine 2013;24:496–502.

Hematological

• Paroxysmal nocturnal hemoglobinuria (PNH)

• Hemolytic uremic syndrome (aHUS and STEC-HUS)

• Catastrophic antiphospholipid antibody syndrome (CAPS)

• Cold agglutinin disease (CAD)

• Autoimmune haemolytic anaemia (AIHA)

• Thrombotic thrombocytopenic purpura (TTP)

Non-hematological

• Dense deposit disease (DDD)

• Age-related macular degeneration (AMD)

• Myocardial infarction

• Sepsis, ARDS

• Systemic lupus erythematosus

• HELLP syndrome

• Rheumatoid arthritis

• Antibody-mediated rejection

• Guillain–Barré syndrome

• Pemphigus

Treated (among others) using plasmapheresis

Take home message

With RBC-concentrates no problems are expected.

Thrombocyte productsThere is no hard evidence that plasma indeed exacerbates• complement activation• induces breakthrough during treatment with anti-C5

…. in complement-mediated diseases, e.g. PNH

Based on in-vitro experiments and theoretical concepts one may advice to prevent minor incompatibility (use then product with PAS or concentrated product)

Plasma products: - if possible avoid in PNH (use e.g. fbg/PCC products)- consider additional dose complement inhibitor

Plasma as a chronic therapy in complement-mediated diseases (aHUS)• Induces responses (complete and partial) but does not prevent ESRD• Plasmapheresis may induce complement activaton• Plasma products contain complement activation products (clinical

relevance?)