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Title: The impact of common variants in C3 and CR1 on C3-mediated extravascular haemolysis in Paroxysmal Nocturnal Haemoglobinuria
Authors: Baral, April Joy
Issue Date: 2023
Publisher: Newcastle University
Abstract: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare haematological disorder characterised by complement-mediated intravascular haemolysis (IVH) due to the loss of CD59 on PNH erythrocytes (E). IVH is inhibited by the anti-C5 antibody, eculizumab but ~30% of patients still require blood transfusions. This is associated with C3 fragment accumulation on PNH-E due to the loss of CD55, causing extravascular haemolysis (EVH) by erythrophagocytosis. C3b accumulation and inactivation on PNH-E are regulated by binding of complement receptor type 1 (CR1) to C3b with factor I (FI) forming a regulatory trimolecular complex (TMC), C3b:CR1:FI. We hypothesised that polymorphisms in C3 and CR1 could influence C3b/iC3b inactivation and can dictate the risk for C3-mediated EVH. The percent of PNH-E loaded with C3 was analysed in eighty-one eculizumab-treated patients that were genotyped for their C3(S/F) and CR1 density (high/low;H/L) polymorphism and a trend for higher C3 loading was observed in patients with C3-S and CR1-L alleles. To investigate this further, recombinant CR1 constructs were generated containing different functional domains (CR11-4, CR18-11, CR11-11) and their interaction with C3b variant proteins and convertases was analysed. Using surface plasmon resonance, CR18-11 and CR11-11 bound more weakly to C3b-S and generated lower levels of TMC, but accelerated decay of convertases was unaltered. Using a novel functional assay, the rate of C3b/iC3b to C3dg conversion mediated by CR1- containing cell lysates with FI was found to be slower from lysates derived from erythrocytes of CR1-H/L donors compared to H/H donors. These data support the hypothesis that the C3-S and CR1-L polymorphisms are potential risk factors for C3-mediated EVH. This may be due to weaker binding of C3b-S to CR1, decreased TMC formation and slower conversion of iC3b to C3dg. We demonstrate that mechanisms leading to increased C3 loading and decreased C3b inactivation may enhance erythrophagocytosis in PNH.
Description: PhD Thesis
Appears in Collections:Translational and Clinical Research Institute

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