Sacha Zeerleder, MD PhD Internist-hematologist Academic Medical Centre, Amsterdam Sanquin Research, Amsterdam

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1 Sacha Zeerleder, MD PhD Internist-hematologist Academic Medical Centre, Amsterdam Sanquin Research, Amsterdam

2 Sacha Zeerleder, MD PhD Internist-hematologist Academic Medical Centre, Amsterdam Sanquin Research, Amsterdam

3 Plasma-containing blood products in Complementmediated diseases beneficial or harmful?

4 Complement system Lectin pathway C4bC2a Alternative pathway C3 Classical pathway C4bC2a Low-grade hydrolysis Inflammation anaphylatoxins (C3a, C5a) C5b Opsonization (, C4b) MAC (C5b-9)

5 Complement system Classical pathway C3 Lectin pathway FI CA P Bb Bb P D DAA B B C5b Decay accelerating activity (DAA) H 2 O for C3 convertase (B/) Factor H (FH) DAF (CD55) Cofactor activity for Factor I (FI): inactivates Factor H (FH) Membrane cofactor protein (MCP) MAC (C5b-9) Wouters & Zeerleder, Haematologica 2015

6 Complement system Classical pathway C3 Lectin pathway FI CA Bb Non-activating FI FH B i DAA B C5b B Equal affinity for FH and B B [2.2uM] FH [3.2uM] MAC (C5b-9) activating Fearon et al ; Law et al. 1977; Volanakis 1998 Wouters & Zeerleder, Haematologica 2015

7 Complement system- amplification loop

8 Amplification loop- regulation

9 Three complement activation pathways: regulation scr1 Lectin pathway Classical pathway C4bC2a C3 C4bC2a C1-inhibitor C4BP MCP (CD46) DAF (CD55) Alternative pathway Inflammation anaphylatoxins (C3a, C5a) Factor I Opsonization (, C4b) Factor H protectin (CD59) C5b MAC (C5b-9) Membrane inhibitors Wouters & Zeerleder, Haematologica 2015 Plasma inhibitors

10 Complement- mediated diseases and hemolysis Lectin pathway C4bC2a C3 Classical pathway C4bC2a Autoimmune Hemolytic Anemia (AIHA) Cold Agglutinin Disease Alternative pathway Inflammation anaphylatoxins (C3a, C5a) Paroxysmal Nocturnal Hemoglobinuria (PNH) Dysregulation alternative pathway loss of control C5b Opsonization (, C4b) Atypical Hemolytic Syndrome (ahus) MAC (C5b-9) Wouters & Zeerleder, Haematologica 2015

11 PNH

12 PNH: deficiency of membrane-bound regulators Lectin pathway C4bC2a Alternative pathway C3 C5b MAC (C5b-9) Classical pathway C4bC2a MCP (CD46) Inflammation DAF (CD55) anaphylatoxins (C3a, C5a) protectin (CD59) Opsonization (, C4b) Membrane inhibitors CD alternative name function CD 14 CD 16 CD 24 CD 48 CD 52 CD 55 CD 59 Pattern recognition receptor (PRR) Fc-gamma receptor IIIb Heat stable antigen Signaling lymphocyte activation molecule 2 (SLAMF2) Campath-1 Inhibition formation C3- convertase Membrane inhibitor of reactive lysis Receptor for LPS Low-affinity receptor IgG Cell adhesion molecule Member Igsuperfamily Not entirely clear Cell-adhesion Inhibition C9 polymerization (MAC) CD 66 CEA family Cell-adhesion Wouters & Zeerleder, Haematologica 2015; Zeerleder, van Solingen & v. Wijk 2015

13 Membrane bound regulators are GPI- linked Zeerleder, van Solingen & v. Wijk 2015 GPI-linked protein Etn Man Man Man Gluc C5b MAC In PIGA -gen

14 The dark side of complement activation and cell-free heme PNH patients with hemolytic disease (n=29) NO consumption (12-fold) PAP 41% (Echo) RV function 80% (MRI) 60% PE 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

15 Inhibition of complement-mediated hemolysis: anti-c5 Eculizumab Humanized monoclonal antibody Blocks cleavage of C5 - decrease in C5a - decrease in C5b9 Anti- C5 Hillmen et al. 2004, Hillmen et al. 2007, Rother et al. 2007, Mache et al 2009, Legendre et al. 2013

16 Exacerbation complement activation in PNH Classical pathway C3 Lectin pathway P Bb P D B C5b Exacerbation due to: Infection Pregnancy Surgery Trauma MAC (C5b-9) Plasma-containing blood products? Roth et al. Int J Hematol. 2011; Kelly et al. NEJM 2015 ; Wouters & Zeerleder, Haematologica 2015

17 How to deal with plasma-containing products in PNH? Amount of plasma per unit 5-10 ml Washed: 0 ml Apheresis: ml PAS III/E: ~127ml/~110ml concentrated: <5 ml 200 ml Jackson et al. J Clin pathol 1992; Rosse, transfusion 1989; Brecher & Taswell; Transfusion 1989 Kelly et al. N Eng J Med 2015; Richtlijn PNH NVH, 2016

18 75 pregnancies in 61 PNH patients Platelet transfusions in 16 pregnancies 2 post-partum hemorrhages complicated by thromboembolic events (1 after plasma infusion)

19 Transfusion of plasma-containing products in PNH Mayo Clinic ( ) 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 salinewashed red cells, 5 units frozen RBCs and 6 units via intraoperative salvage) Brecher & Taswell; Transfusion episode of postransfusion hemolysis (O product to a AB recipient)

20 1978: VSAA, treatment with ATG; no PNH 1980: 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 insufficiency Diagnosis PNH (positive Ham- and sucrose lysis test) Plasma donors (n=4) O neg (no irregular antibodies) In-vitro Induces lysis of A1 Test RBCs (titer 1:2) Induces lysis of A1 patient RBCs (titer 1:512)

21 Minor incompatibility: exacerbation complement activation Anti-A1 Classical pathway C3 A1 CP activation P D P Bb B C5b Roth et al. Int J Hematol. 2011; Kelly et al. NEJM 2015 ; Wouters & Zeerleder, Haematologica 2015 MAC (C5b-9)

22 How to deal with plasma-containing products in PNH? Amount of plasma per unit 5-10 ml Washed: 0 ml No washing needed Apheresis: ml PAS III/E: ~127ml/~110ml concentrated: <5 ml Minor mismatch PAS III/E, concentrated product 200 ml If possible: avoid Give additional dose eculizumab Jackson et al. J Clin pathol 1992; Rosse, transfusion 1989; Brecher & Taswell; Transfusion 1989 Kelly et al. N Eng J Med 2015; Richtlijn PNH NVH, 2016

23 Atypical HUS

24 Thrombotic microangiopathy (TMA) - ahus Hemolytic anemia Thrombocytopenia Fever Neurological symptoms Renal dysfunction ADAMTS13 Complement activation/-dysregulation Endothelilal destruction (Shiga toxine) UL WF C5C9b

25 Atypical HUS: complement regulation on tilt Classical pathway C3 Lectin pathway FI CA P Bb Bb P D DAA B B C5b Decay accelerating activity (DAA) H 2 O for C3 convertase (B/) Factor H (FH) Inflammation DAF (CD55) anaphylatoxins Cofactor activity for Factor I (FI): (C3a, C5a) inactivates Factor H (FH) Membrane Opsonization cofactor protein (MCP - CD46) (, C4b) Wouters D, Zeerleder S. Haematologica. 2015;100(11): MAC complex (C5b-9)

26 Atypical HUS : Complement Factor H (CFH) Complement Control Protein repeats: CCP repeats (à 60 aa) Mw~150 kd Chromosome 1q32 Mutations ahus: 30% mutations in FH 20% 60% NH COO Recognition-site : dissociation C3-convertase (B, ) Cofactor for CFI (cleavage ) Recognition-site surfaces: Basement membranes Endothelium Autoantibodies to FH Kavanagah & Goodship Nephron Clin Pract.2010, Noris & Remuzzi, N Engl J Med 2009, Richards et al. Adv Immunol 2007

27 ahus: complement regulation alternate pathway versus C5 CFH -/- del16-20: Spontaneous development of ahus No C5 activation - no damage Pickering et al. J Exp Med 2007 de Jorge et al. J Am Soc Nephrol 2011

28 FH mutation renal damage complement deposition No complement deposition

29 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 H Kavanagah & Goodship. Nephron Clin Pract. 2011;118(1):c37-42, Noris & Remuzzi, N Engl J Med 2009;361(17): Richards et al. Adv Immunol 2007; 96: Lemaire et al. Nat Genet. 2013;45(5):531-6.

30 End-stage renal failure/death in patients with ahus MCP No mutation Auto-AB CFH CFI C3 CFH Marina Noris et al. CJASN 2010;5:

31 ahus mutations and response to plasma Mutated gene/protein CFH (incl. CFH/CFHR1 hybrid genes) type Frequency (%) Death/ESRD (%) Response to plasma (%) LCR % MCP (CD46) LCR 5-9 <20 97% CFI LCR % C3 GoF % CFB GoF Thrombomodulin? % CHFR1/3 def. with anti-fh antibodies LCR % Diacylglycerol kinase ɛ Prothrom none % Plasmapheresis: aim to restore regulators and/or remove dysfunctional protein 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

32 ahus: effects dialysis and plasmapheresis on complement activation before after c p< before after C4bc p<0.001 ahus patients hemodialysis (n= 6) Eculizumab (n= 2) Plasmapheresis (n=3) man woman nm 100 nm 400 Median age 52,3 25, dialysis anti-c5 PF dialysis anti-c5 PF nm /c (normal <57 nm) before C4b/c (normal < 8 nm) after nm normal <57 nm c normal <8 nm C4bc 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

33 Anti-C5 (Eculizumab) treatment in ahus Platelet counts Renal function

34 Uncontrolled Complement Activation Has a Role in Many Diseases Hematological Non-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) 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 1. Schrezenmeier H. Transfus Apher Sci. 2012;46:87-92; 2. Holers VM et al. Immunol Rev. 2008;223: Huda R. Rev Neurosci. 2014; doi.10.15/14. Epub ahead of print. 4. Meri S. European Journal of Internal Medicine 2013;24:

35 Take home message With RBC-concentrates no problems are expected. Thrombocyte products There 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?)

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