Elevated plasma Complement Factor H Regulating Protein 5 is associated with venous thromboembolism and COVID-19 severity

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Venous thromboembolism (VTE), comprising both deep vein thrombosis (DVT) and pulmonary embolism (PE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. We used multiplex proteomics profiling to screen plasma from patients with suspected acute VTE, and a case-control study of patients followed up after ending anticoagulant treatment for a first VTE. With replication in 5 independent studies, together totalling 1137 patients and 1272 controls, we identify Complement Factor H Related Protein (CFHR5), a regulator of the alternative pathway of complement activation, as a novel VTE associated plasma biomarker. Using GWAS analysis of 2967 individuals we identified a genome-wide significant pQTL signal on chr1q31.3 associated with CFHR5 levels. We showed that higher CFHR5 levels are associated with increased thrombin generation in patient plasma and that recombinant CFHR5 enhances platelet activation in vitro . Thrombotic complications are a frequent feature of COVID-19; in hospitalised patients we found CFHR5 levels at baseline were associated with short-time prognosis of disease severity, defined as maximum level of respiratory support needed during hospital stay. Our results indicate a clinically important role for regulation of the alternative pathway of complement activation in the pathogenesis of VTE and pulmonary complications in acute COVID-19. Thus, CFHR5 is a potential diagnostic and/or risk predictive plasma biomarker reflecting underlying pathology in VTE and acute COVID-19.

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  1. SciScore for 10.1101/2022.04.20.22274046: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsConsent: Exclusion criteria were patients with on-going anticoagulant treatment, pregnancy, active cancer, short life expectancy or lack of capacity to leave approved consent.
    IRB: Approval for VEBIOS was granted by the regional research ethics committee in Stockholm, Sweden (KI 2010/636-31/4) and all participants gave informed written consent, in accordance with the Declaration of Helsinki.
    Sex as a biological variable, alcohol consumption and smoking habits; (3) family history of VTE (4) ongoing antithrombotic (antiplatelet) treatment and (5) estrogen containing contraceptives and hormone replacement therapy (women only).
    RandomizationBriefly, paired samples were randomly distributed within the same 96-well area.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Plasma proteomic profiles in VEBIOS ER were generated using multiplexed suspension bead arrays (SBA) with 758 antibodies selected from the Human Protein Atlas (HPA) project, targeting 408 proteins (Table S1), using identical design, procedures and methods as previously described [27].
    suggested: None
    Immunocapture mass spectrometry (IC-MS): IC-MS was performed in triplicate, as previously described [27] using the HPA059937 antibody (Atlas Antibodies) or rabbit immunoglobulin G (rIgG) as a negative control.
    suggested: None
    rabbit immunoglobulin G ( rIgG
    suggested: None
    In-house developed bead based dual binder immunoassays: A Suspension Bead Array (SBA) was built with the capture antibodies raised against human extracellular sulfatase 1-SULF1 (rabbit polyclonal HPA059937) and human CFHR5 (rabbit polyclonal HPA072446 and HPA073894) covalently coupled to color-coded magnetic beads as previously described (Drobin et al., 2013; Neiman et al., 2013).
    human extracellular sulfatase 1-SULF1
    suggested: None
    suggested: None
    Mouse anti-human SULF1 antibodies Abnova ABIN525031, Abcam ab172404, Thermofisher PA5-113112, Human Protein Atlas antibodies HPA054728 and HPA051204, and mouse monoclonal anti-human CFHR5 (R&D systems, MAB3845) antibody were labelled with biotin and used as detection antibodies in combination with their respective capture antibodiesCitrate plasma samples were thawed on ice and centrifuged for 1 min at 2000 rpm and diluted in buffer polyvinyl casein 10% rIgG (PVXcas 10% rIgG; polyvinyl alcohol, Sigma Aldrich P8136; polyvinylpyrrolidone
    Mouse anti-human SULF1 antibodies
    suggested: None
    anti-human SULF1
    suggested: (Abnova Cat# MAB3845, RRID:AB_10628528)
    Human Protein Atlas antibodies
    suggested: (Atlas Antibodies Cat# HPA054728, RRID:AB_2682586)
    suggested: None
    Absolute quantification of CFHR5, C3 and D-dimer: For CFHR5 quantification, rabbit polyclonal anti-human CFHR5 HPA072446 (Atlas Antibodies) and mouse monoclonal anti-human CFHR5 (R&D systems; MAB3845) antibodies were used in a dual binder assay.
    suggested: None
    anti-human CFHR5
    suggested: None
    For C3 quantification, mouse anti-human C3 and mouse monoclonal anti-human C3 antibodies (Bsi0263, Bsi0190, respectively, Biosystems International) were used in a dual binder assay.
    anti-human C3
    suggested: None
    Platelets were subsequently incubated with primary antibodies: anti-human CD62P-AF647 (AK4), anti-human CD63-PE (H5C6) or anti-human CD41/CD61-FITC (PAC-1) (all Biolegend) for 20 minutes, washed (PBS then 500 g for 10 minutes), then fixed with 1 % paraformaldehyde and incubated with Alexa Fluor 647-streptavidin (Jackson Immuno Research, Ely, UK) for 20 minutes.
    anti-human CD62P-AF647
    suggested: None
    suggested: None
    anti-human CD63-PE
    suggested: None
    anti-human CD41/CD61-FITC
    suggested: None
    suggested: None
    Software and Algorithms
    Uniprot complete human proteome (update 20180131) was used to query the raw data, with the engine Sequest and Proteome Discoverer platform (PD, v1.4.0.339, Thermo Scientific).
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    Absolute quantification of CFHR5, C3 and D-dimer: For CFHR5 quantification, rabbit polyclonal anti-human CFHR5 HPA072446 (Atlas Antibodies) and mouse monoclonal anti-human CFHR5 (R&D systems; MAB3845) antibodies were used in a dual binder assay.
    R&D systems
    suggested: None
    Remaining polymorphisms were then imputed using the TOPMed r2 reference panel using Eagle v2.4.
    suggested: (Eagle, RRID:SCR_017262)
    A control quality has been performed on individuals and genetic variants using Plink v1.9 and the R software v3.6.2 (Chang et al., 2015).
    suggested: (PLINK, RRID:SCR_001757)
    Results obtained in the different contributing cohorts were then meta-analyzed through a random effect model as implemented in the GWAMA software [91].
    suggested: (GWAMA, RRID:SCR_006624)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Our study has various strength and limitations; VEBIOS ER, the discovery cohort, that was derived from a single centre, where blood sampling for plasma biobanking was performed in parallel to that for routine tests after initial evaluation (before diagnostic imaging or anticoagulant treatment), thus avoiding bias in inclusion or biobanking. Samples were handled according to standard clinical chemistry lab routine, thus variations in needle-to-spin-to-freeze time were equivalent between case and control samples. As biobanking was based on the routine sample flow, this increases the feasibility that identified biomarker candidates are suitable for clinical translation into a routine setting. Importantly, we demonstrate an association of CFHR5 with VTE in several independent studies, that include patients in the acute setting, at follow up, and prior to recurrence. One limitation of our study is that we have not analysed a cohort of individuals that were sampled prior to VTE event. From a technological perspective, our study demonstrates the need for orthogonal verification of any potential biomarker identified using antibody-based proteomics screening [34, 35]. The same caution should be extended to findings generated using other high throughput affinity proteomics technologies vulnerable to non-specific protein binding, such as aptamer-based [74], where missense single nucleotide polymorphisms can affect binding in a manner where a genetic difference drive associations, rather...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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