PMC9204634_01
Timeline of hospital course of patient after a living unrelated kidney transplantation. C3, complement 3; HD, hemodialysis; LDH, lactate dehydrogenase; OR, operative room; thymo, thymoglobulin; MPA, mycophenolic acid.
fimmu-13-909503-g001.jpg
PMC9204634_01_fimmu-13-909503-g001.jpg
f1
Laboratory data were notable for anemia (hemoglobin 6.9-7.7 g/dL; reference range 13-17 g/dL), severe thrombocytopenia (platelet count 24-36 k/microL; reference range 150-400 k/microL), low haptoglobin (<10 mg/dL; reference range 30-200 mg/dL) and high lactate dehydrogenase (580-736 units/L; reference range 150-250 units/L) (  Figure 1  ).
Biopsy image from patient. Fibrin thrombi seen in glomeruli (black arrow). Small arteries and arterioles demonstrated focal fibrinoid necrosis of the arterial wall (not shown).
fimmu-13-909503-g002.jpg
PMC9204634_01_fimmu-13-909503-g002.jpg
f2
POD 3. Patient returned to the OR on POD 4 for revision and closure and at that time an intraoperative biopsy was performed which confirmed a TMA with no evidence of acute cellular or antibody-mediated rejection ( Figure 2 ).
Functional evaluation of Factor I (FI) variant Ile357Met: proteolytic activity. The fluid-phase C3b proteolytic activity of the variant factor I (357Met) with its cofactor proteins (Factor H [FH], membrane cofactor protein [MCP], or complement receptor 1 [CR1]) was assessed by cleavage of purified C3b to iC3b and compared to wild type (WT). For these assays, purified WT and FI variant proteins were diluted in physiologic salt (150 mM NaCl) buffer with C3b (10 ng; Complement Technologies, Inc, Tyler, TX USA) at 37 C. Concentrations of WT or variant FI used with the individual cofactors were 10 ng with MCP, 20 ng with FH and 15 ng with CR1. Concentration of cofactor used in the reactions were 100 ng MCP, 200 ng FH or 150 ng CR1. Reactions were carried out in a total volume of 15 microl/reaction at 37 C. Kinetic analysis of the WT and variants was achieved through collection of sample at 0, 10, 20 and 30 min. At each time point 7 microl of 3x Laemmli reducing sample buffer was added to individual reactions to stop the reaction and then heated at 95 C for 5 min. The samples were electrophoresed on 10% Tris-glycine gel and then transferred to nitrocellulose for WB analysis. Membranes were rinsed with TBS-T (0.05%Tween-20) for 5 min and blocked overnight with 5% nonfat dry milk in PBS. Blots were probed with a 1:5,000 dilution of goat anti-human C3 (Complement Technologies, Inc, Tyler, TX, USA) followed by HRP-conjugated rabbit anti-goat IgG and developed with SuperSignal substrate (Thermo Fisher Scientific, Waltham, MA, USA). The signal detected on radiographic films was scanned using a laser densitometer (Pharmacia LKB Biotechnology, Piscataway, NJ, USA). Multiple exposures were used to establish linearity. (A, B). The percentage of alpha' chain remaining and generation of alpha41 fragment indicates cleavage of C3b to iC3b. Cleavage rate was measured by densitometric analysis of the alpha' chain remaining as well as generation of alpha41 relative to the beta chain. Data represent 2 separate experiments with bars corresponding to the standard error of mean (SEM). Upon comparison to WT FI, the proteolytic activity of variant 357Met was defective with FH. The P value for the difference in the percentage of alpha'chain remaining between WT and variant was 0.05 and for the difference in the percentage of alpha41 generation was <0.05. (C). Representative WB demonstrating cofactor activity of Factor H with the variant (357Met) compared with wild type FI as well as purified FI (D) No defect was observed with MCP or CR1 as the cofactor protein. I, isoleucine; M, methionine. Structural evaluation of Ile357Met (E) Mapping of Ile357 on the structure of FI shows that it is located in the serine protease domain of FI which harbors the catalytic activity. Although the variant is away from the catalytic serine (S525)), the substitution of I to a bulkier amino acid M likely alters the position of the disulfide bond 365-381, thus affecting the FI activity. (F) Mapping of the Ile357 on the triple complex of Factor H and C3b shows that the variant is away from the binding surface of FH, therefore we speculate that it likely leads to a conformational change resulting in low functional activity.
fimmu-13-909503-g003.jpg
PMC9204634_01_fimmu-13-909503-g003.jpg
f3
Additionally, functional analysis demonstrated that the variant had defective complement regulatory activity with Factor H ( Figures 3A- C ) but no defect was seen with membrane cofactor protein or complement receptor 1 ( Figure 3D ). The disulfide bond plays a vital role of keeping the catalytic histidine (H380) in place for optimal enzymatic activity ( Figure 3E ). We also mapped the variant on the triple complex with FH and C3b ( Figure 3F ) and it does not seem to lay close to FH, therefore, we speculate that the variant likely causes a conformational change that results in reduced functional activity.