Journal: Nature Communications

Article Title: Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism

doi: 10.1038/s41467-023-38383-y

Figure Lengend Snippet: a Overview of the VEBIOS ER discovery cohort. 756 HPA antibodies, targeting 408 candidate proteins, were used to analyse plasma samples using affinity proteomics. Log fold changes in antibody MFI (median fluorescent intensity) signal were calculated between VTE cases ( n = 48) and controls ( n = 48) in ( b ) citrate or ( c ) EDTA anticoagulated plasma; coloured circles indicate antibodies that generated signals significantly associated with VTE in both. MFI signals generated by these antibodies for controls ( n = 48) and cases ( n = 48) in ( d ) citrate plasma and ( e ) EDTA plasma. f Immunocapture-mass spectrometry identification of protein targets of HPA059937 ( n = 3 of independent biological replicates). g Dual binder assays were developed using an anti-CFHR5 detection antibody, combined with HPA059937 (raised against SULF1), anti-CFHR5 HPA073894 or anti-CFHR5 HPA072446 as capture antibodies. Monoclonal anti-CFHR5 (MAB3845) was applied as detection antibody in the three combinations. CFHR5 levels in the citrated plasma samples were re-analysed, using the respective dual binder assays, to determine levels (MFI) in controls ( n = 48) vs . cases ( n = 48) and the correlation between the signal and those generated by the original single binder assay using HPA059937 [all p < 1E-04]. Dual binder assay using capture antibody HPA072446 with a recombinant protein standard and MAB3845 as detection antibody, was used for absolute quantification of CFHR5 in samples from: ( h ) VEBIOS ER and ( i ) VEBIOS Coagulation (controls = 135, cases = 142). CFHR5 concentration was measured in controls and cases, with associated OR (odds ratio per 1 standard deviation increase [ h and i , left]) or used to determine the correlation with C-reactive protein (CRP), or D-dimer concentration [ h and i , right panels]. All data in dot plots (1 d – i ) are represented as median value with 95% CI (confidence interval). Case and control groups was compared using a linear model adjusting for age and sex in d , e , g , h and i .***** p < 0.00001, **** p < 0.0001, *** p < 0.001, ** p < 0.01. Two-sided Spearman´s correlation analysis for h and i . For summary statistics see Supplementary data 1 [Tab_2, Panel A] . Source data are provided as a Source Data file ( b – i ).

Article Snippet: The polyclonal anti-human CFHR5 HPA072446 capture antibody and the anti-human CFHR5 MAB3845 detection antibody were selected for development of a quantitative assay together with human recombinant CFHR5 (R&D systems; 3845-F5).

Techniques: Clinical Proteomics, Generated, Mass Spectrometry, Recombinant, Quantitative Proteomics, Coagulation, Concentration Assay, Standard Deviation, Control

Journal: Nature Communications

Article Title: Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism

doi: 10.1038/s41467-023-38383-y

Figure Lengend Snippet: Characteristics of VEBIOS ER case control discovery study-blood concentrations

Article Snippet: The polyclonal anti-human CFHR5 HPA072446 capture antibody and the anti-human CFHR5 MAB3845 detection antibody were selected for development of a quantitative assay together with human recombinant CFHR5 (R&D systems; 3845-F5).

Techniques: Control, Concentration Assay

Journal: Nature Communications

Article Title: Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism

doi: 10.1038/s41467-023-38383-y

Figure Lengend Snippet: Association of CFHR5 concentration and VTE

Article Snippet: The polyclonal anti-human CFHR5 HPA072446 capture antibody and the anti-human CFHR5 MAB3845 detection antibody were selected for development of a quantitative assay together with human recombinant CFHR5 (R&D systems; 3845-F5).

Techniques: Concentration Assay

Journal: Nature Communications

Article Title: Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism

doi: 10.1038/s41467-023-38383-y

Figure Lengend Snippet: a mRNA expression of CFHR5 across 55 different human tissue types. b Expression of CFHR5 in different liver cell types, analysed by ssRNAseq. c STRING protein-protein interaction analysis for genes identified as potentially co-expressed with CFHR5 in liver by correlation-based analysis of bulk mRNAseq. Complement component 3 (C3) concentration was measured in plasma from ( d ) VEBIOS ER ( n = 48 + 48) or ( e ) VEBIOS coagulation ( n = 135 + 142) to determine: differences between controls and cases ( d and e , left), or correlation with CFHR5 in controls or cases ( d and e , right panels). Case and control groups was compared using a linear model adjusting for age and sex ( d , e ) ** p < 0.01. Dot plots ( d , e ) are represented as median value with 95% CI. Two-sided Spearman´s correlation analysis for 2 d and 2 e . Summary of statistical analysis can be found in Supplementary data 1 [Tab_2, Panel B] . Source data are provided as a Source Data file ( d , e ). C4BPA complement factor 4 binding protein, CFI complement factor I, CFB complement factor B, CFH complement factor H, C1S complement component 1, C1R complement component 1, C2 complement component 2, C8a complement component C8 alpha chain, C8b complement component C8 beta chain, C5 complement component 5, C9 complement component 9.

Article Snippet: The polyclonal anti-human CFHR5 HPA072446 capture antibody and the anti-human CFHR5 MAB3845 detection antibody were selected for development of a quantitative assay together with human recombinant CFHR5 (R&D systems; 3845-F5).

Techniques: Expressing, Concentration Assay, Clinical Proteomics, Coagulation, Control, Binding Assay

Journal: Nature Communications

Article Title: Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism

doi: 10.1038/s41467-023-38383-y

Figure Lengend Snippet: Plasma samples were generated as part of: ( a ) the Swedish VEBIOS ER or ( b ) the Swedish DFW-VTE study, both of which recruited patients presenting with suspected VTE. Samples were drawn pre- treatment, and cases and controls were identified based on confirmed or ruled out diagnosis. c The French FARIVE study recruited patients with confirmed acute VTE, with controls recruited from hospital patients treated for non-VTE causes. Samples were drawn within 1 week from diagnosis, during initiation of treatment ( d ) The Swedish VEBIOS Coagulation or ( e ), Spanish RETROVE study recruited cases from patients who had a prior first time VTE, sampled post-treatment (6–12 months anticoagulants), with healthy controls recruited from the general population. CFHR5 concentration was measured in the respective samples using a dual binder assay. Case and control groups was compared using a linear model adjusting for age and sex (3 a–e ). *** p < 0.001, **** p < 0.0001. OR (1 SD) = Odds ratio for 1 standard deviation elevation. CI confidence interval. All dot plots are represented as median value with 95% CI. Source data are provided as a Source Data file.

Article Snippet: The polyclonal anti-human CFHR5 HPA072446 capture antibody and the anti-human CFHR5 MAB3845 detection antibody were selected for development of a quantitative assay together with human recombinant CFHR5 (R&D systems; 3845-F5).

Techniques: Clinical Proteomics, Generated, Biomarker Discovery, Coagulation, Concentration Assay, Control, Standard Deviation

Journal: Nature Communications

Article Title: Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism

doi: 10.1038/s41467-023-38383-y

Figure Lengend Snippet: a Manhattan plot of the meta-analysis on INVENT-MVP consortium resources [17] showing six loci associated with CHFR5 plasma levels and VTE risk: CFHR1, CFHR4 (rs10737681, p = 2.94E-396), HNF1A (rs2393776, p = 1.48E-21), JMJD1C (rs7916868, p = 4.61E-12), TRIB1 (rs28601761, p = 4.39E-09), DNAH10 (rs7133378, p = 2.43E-08) and HNF4A (rs1800961, p = 4.97E-08). Lead SNPs at HNF1A and DNAH10 are rs2393776 and rs7133378, respectively. They are ~3 Mb apart and do not show any linkage disequilibrium (pairwise r 2 = 0). b Regional association plot at the Chromosome 1 locus covering >10 Mb from CFHR1 to CFHR5 around the lead SNP associated with CFHR5 plasma levels. c CFHR5 plasma levels for 16 patients who are carriers of rare non-synonymous CFHR5 -associated variants (rs139017763, rs41299613 or rs35662416) and non-carriers ( n = 1214). t -test, two sided. ***** p < 0.00001. Dot plot ( c ) is represented as median value with 95% CI. See also Supplementary data 1 [Tabs_11–13] . Source data are provided as a Source Data file ( c ).

Article Snippet: The polyclonal anti-human CFHR5 HPA072446 capture antibody and the anti-human CFHR5 MAB3845 detection antibody were selected for development of a quantitative assay together with human recombinant CFHR5 (R&D systems; 3845-F5).

Techniques: Clinical Proteomics

Journal: Nature Communications

Article Title: Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism

doi: 10.1038/s41467-023-38383-y

Figure Lengend Snippet: Platelet activation was measured by surface expression of ( a ) P-selectin, ( b ) activated GP IIb/IIIa (PAC1 + ) or ( c ) CD63, following treatment of platelet rich plasma with different concentrations of adenosine diphosphate (ADP), convulxin or TRAP6, [ a – c : top, middle and bottom panels, respectively] following pre-incubation with recombinant CFHR5, or PBS control. d Platelet aggregation was measured in response to ADP (2 μm) following pre-incubation with recombinant CFHR5, or PBS control: representative aggregation curve [left], maximum aggregation [middle] and slope [right], of four independent experiments. ADP-induced platelet activated GP IIb/IIIa (PAC1 + ) was measured following preincubation with: ( e ) DMSO (control) or compstatin, or ( f ) PBS (control) or an anti-C3a antibody, followed by treatment with PBS or rCFHR5. US: unstimulated (PBS control). Each experiment is represented by an individual point and paired experiments connected by a dotted line. Anova ( a – c ) and t- test ( d – f ) were performed (two-tailed). * p < 0.05 ** p < 0.01 *** p < 0.001 ( p for trend bottom right). Source data are provided as a Source Data file (Fig. 5).

Article Snippet: The polyclonal anti-human CFHR5 HPA072446 capture antibody and the anti-human CFHR5 MAB3845 detection antibody were selected for development of a quantitative assay together with human recombinant CFHR5 (R&D systems; 3845-F5).

Techniques: Activation Assay, Expressing, Clinical Proteomics, Incubation, Recombinant, Control, Two Tailed Test

Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Glomerular FHR5 staining in C3 glomerulopathy. (a) Glomerular staining intensity in native and transplant biopsies. FHR5 was the most frequent protein at 3+ intensity and detected in all of the transplant biopsies. (b) Representative images of complement staining in the native kidney. For each set of images, the biopsy indication, the appearances of the glomeruli by light microscopy, and the estimated glomerular filtration rate (eGFR) and proteinuria at the time of biopsy are indicated. *The cases represented by the fourth and fifth rows of images are from the same family. (c) Representative images of complement staining in transplant kidneys. For each set of images, the biopsy indication (either protocol or due to clinical changes; i.e., indication biopsy), time posttransplant together with the eGFR, and proteinuria at the time of biopsy are indicated. The histology including the presence or absence of rejection and the histology of subsequent biopsies is also shown. C3G, C3 glomerulopathy; C3GN, C3 glomerulonephritis; EH, endocapillary hypercellularity; fH, factor H; FHR1, factor H–related protein 1; FHR5, factor H–related protein 5; FHR5 mutation, the mutation described in CFHR5 nephropathy ; MPGN, membranoproliferative glomerulonephritis; NS, nephrotic syndrome; UPCR, urinary protein:creatinine ratio. Original magnification ×400. Bars = 100 μm.

Article Snippet: FHR5 staining was eliminated after pre-incubation of the primary antibody with purified FHR5 (#3845-F5; R&D Systems, Minneapolis, MN; ).

Techniques: Staining, Light Microscopy, Filtration, Mutagenesis

Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Sequential glomerular FHR5 staining in a single case of C3 glomerulopathy transplant recurrence. Images and information from the same biopsy are organized in columns. A protocol surveillance biopsy performed 3 months after transplantation (Transplant biopsy 1) showed recurrent C3 glomerulonephritis (C3GN) with mild endocapillary hypercellularity (EH). Glomerular factor H–related protein 5 (FHR5), C5b9, C3b/iC3b/C3c, and C3dg were detected. The estimated glomerular filtration rate (eGFR) was stable and there was no significant proteinuria. Approximately 6 months posttransplant, the patient developed proteinuria and a fall in eGFR. Biopsy showed crescentic C3GN and increased glomerular CD68-positive cells (Transplant biopsy 2). Glomerular FHR5, C5b9, C3b/iC3b/C3c, and C3dg were detected but at increased staining intensity compared with the first transplant biopsy. The eGFR and proteinuria improved with eculizumab and prednisolone treatment. After 4 months of eculizumab treatment, renal biopsy (Transplant biopsy 3) showed resolution of glomerular CD68 staining, but glomerular staining for FHR5, C5b9, C3b/iC3b/C3c, and C3dg remained unchanged. After a further 3 months, proteinuria increased and biopsy showed crescentic C3GN and the recurrence of glomerular CD68-positive cells (Transplant biopsy 4). Proteinuria improved with re-introduction of eculizumab. UPCR, urine protein:creatinine ratio. Original magnification ×200 or ×400. Bars = 100 μm.

Article Snippet: FHR5 staining was eliminated after pre-incubation of the primary antibody with purified FHR5 (#3845-F5; R&D Systems, Minneapolis, MN; ).

Techniques: Staining, Transplantation Assay, Filtration

Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Glomerular factor H–related protein 5 (FHR5) colocalizes with C3 in C3 glomerulopathy. (a) Glomerular FHR5 staining intensity positively correlated with the staining intensity of C3b/iC3b/C3c, C3dg, and C5b9. Both glomerular C3b/iC3b/C3c and C3dg positively correlated with glomerular C5b9. R values are calculated from Pearson’s correlation coefficient and P values are adjusted for multiple comparisons. (b) Representative images of combined immunofluorescence staining in three C3G cases for glomerular FHR5 with either C3b/iC3b/C3c or C3dg, and C3b/iC3b/C3c with C3dg. Renal biopsies in all 3 cases showed C3-dominant membranoproliferative glomerulonephritis, and the biopsy indications together with the urine protein:creatinine ratio (UPCR), estimated glomerular filtration rate (eGFR), and serum C3 levels at the time of biopsy are listed. The staining patterns for C3b/iC3b/C3c and C3dg (right-hand column of images) showed areas of colocalization (arrows), areas of C3b/iC3b/C3c alone (stars), and areas of C3dg alone (triangles). The staining pattern for C3b/iC3b/C3c and FHR5 (left-hand column of images) showed areas of colocalization (arrows), areas of C3b/iC3b/C3c alone (stars), and areas of FHR5 alone (triangles). The staining pattern for C3dg and FHR5 (middle column of images) showed areas of colocalization, particularly along capillary walls in cases 2 and 3 (arrows) and areas of FHR5 alone in cases 1 and 2 (triangles). Notably, we did not detect areas of C3dg without FHR5 staining. (c) FHR5, C3b/iC3b/C3c, and C3dg glomerular locations correlate in C3G. We calculated the correlation of glomerular antigen locations in all available glomeruli from the three C3G cases (c). The Pearson’s correlation coefficient for all glomeruli from cases 1 (circles), 2 (squares), and 3 (triangles), and the median values of the correlations for each case (horizontal lines) are shown. The median values of the correlation coefficients ( r ) for each case were as follows: C3b/iC3b/C3c with FHR5: 0.73 (case 1), 0.76 (case 2), and 0.68 (case 3); FHR5 with C3dg: 0.71 (case 1), 0.90 (case 2), and 0.5 (case 3); and C3b/iC3b/C3c with FHR5: 0.68 (case 1), 0.81 (case 2), and 0.58 (case 3). MMF, mycophenolate mofetil. Original magnification ×400. Bars = 100 μm.

Article Snippet: FHR5 staining was eliminated after pre-incubation of the primary antibody with purified FHR5 (#3845-F5; R&D Systems, Minneapolis, MN; ).

Techniques: Staining, Immunofluorescence, Filtration

Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Glomerular factor H–related protein 1 (FHR5) and C5b9 staining intensity associated with lower estimated glomerular filtration rate (eGFR) at biopsy in C3 glomerulopathy. The eGFR at the time of biopsy was significantly lower in biopsies that had maximal staining intensities for FHR5 ( P = 0.04, difference of medians 19.7 ml/min per 1.73 m 2 ; 95% confidence interval [CI] 1.1–43.0) and C5b-9 ( P = 0.03, difference of medians 14.86 ml/min per 1.73 m 2 ; 95% CI 3.8–46.6). This was not seen for glomerular factor H–related protein 1 (FHR1), C3b/iC3b/C3c, C3dg, and C4d. P values are derived from Mann-Whitney U tests.

Article Snippet: FHR5 staining was eliminated after pre-incubation of the primary antibody with purified FHR5 (#3845-F5; R&D Systems, Minneapolis, MN; ).

Techniques: Staining, Filtration, Derivative Assay, MANN-WHITNEY

Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Tubulo-interstitial staining for FHR5 in C3 glomerulopathy. (a) Representative images of staining for factor H–related protein 5 (FHR5), factor H–related protein 1 (FHR1), factor H (fH), properdin, and C3b/iC3b/C3c. No tubulo-interstitial staining for FHR5 or fH was evident but there was strong tubulo-interstitial staining for both properdin and FHR1. (b) Tubulo-interstitial staining for properdin was also seen in other renal diseases (thin basement membrane disease [TBM], lupus nephritis [LN], and membranous nephropathy). Tubulo-interstitial staining for FHR1 was demonstrable in biopsies from patients with TBM and IgA nephropathy. Tubulo-interstitial FHR1 staining in TBM was still detectable but less intense when the proteinase XXIV enzyme was used instead of pressure cooker antigen retrieval. Glomerular and tubulo-interstitial FHR1 was absent in renal tissue from a patient with IgA nephropathy and FHR1 deficiency. C3G, C3 glomerulopathy; LN IV(A/C), lupus nephritis class 4, active and chronic. Original magnification ×200. Bars = 100 μm.

Article Snippet: FHR5 staining was eliminated after pre-incubation of the primary antibody with purified FHR5 (#3845-F5; R&D Systems, Minneapolis, MN; ).

Techniques: Staining, Membrane