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We have previously shown that the C3D1115N mouse, engineered around a single point mutation in C3 associated with atypical haemolytic uraemic syndrome (aHUS) in man, fully recapitulates the clinical disease. In this study, we investigated the role of properdin in aHUS. C3D1115N mice were crossed onto properdin-deficient mice or C3D1115N mice were treated with anti-properdin monoclonal antibody therapy and survival tracked. Kidney function, serum biomarkers and kidney pathology was carefully assessed at set end points. C3 and Fibrin deposition was assessed using immuno-fluorescence. We have found that removing properdin ameliorates disease, although two mice had evidence of renal disease over a ~6-month period. Therapeutic treatment with an anti-properdin monoclonal antibody (14E1) after evidence of clinical disease was sufficient to block the progression of disease, suggesting that measures to reduce the stability of the alternative pathway C3 and C5 convertases can help prevent thrombotic microangiopathic anaemia (TMA)/aHUS. Our data indicate that a fine balance exists between disease progression and resolution in our C3 gain-of-function model. The data also suggest that anti-properdin therapy, as noted in another mouse model of TMA, may provide a viable treatment option to maintain remission in complement mediated aHUS.

Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches. Properdin is the only known positive regulator of the human complement system, stabilising the convertase C3 in the alternative pathway of complement activation. Here, the authors report the identification and characterisation of a species-specific properdin inhibitor CirpA, derived from tick saliva.

Aberrant complement activation can cause damage to newly formed fetal-derived structures and excessive inflammatory response at the feto-maternal interface, contributing to pregnancy-related complications, including preeclampsia (PE), which is one of the most severe pathologies in new-borns. Properdin is the only known positive regulator of the complement alternative pathway, as it stabilizes the inherently labile C3bBb complex and amplifies its activity. This study describes the presence of properdin in PE and investigates its role in the pathogenesis. We examined the distribution and expression of properdin at both the transcript and protein levels in term placental tissue, serum, placental syncytiotrophoblast microvesicles (STBMs), and circulating placental exosomes from PE women compared to healthy mothers, using RT-qPCR, western blot, immunohistochemistry, transmission electron microscopy (TEM), and immunofluorescence. To link properdin levels with alternative pathway complement factors, we also assessed the expression of C3 and C5. PE placentae showed significantly higher properdin, C3 and C5 at transcript as well as protein levels compared to healthy placentae. Conversely, properdin levels in serum, STBMs, and circulating placental exosomes were lower in PE compared to healthy pregnancies. Immunohistochemical analysis revealed properdin distribution throughout the PE placentae, with higher concentrations at the syncytial knots containing pyknotic nuclei were observed via TEM, along with elevated levels of cleaved caspase 3. Thus, properdin was significantly upregulated in the PE placentae, along with C3 and C5, and might be associated with the apoptotic nuclei inside syncytial knots. This evidence suggests that properdin may trigger complement-mediated damage to the placental barrier, exacerbating the development of PE placentae.

Properdin is an endogenous positive regulator of the complement alternative pathway (AP). Tarperprumig (ALXN1820), a novel humanized bispecific antibody, binds properdin and albumin and is being developed to treat complement‐mediated diseases. This phase I, randomized, double‐blind, placebo‐controlled trial assessed the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and immunogenicity of tarperprumig in healthy adult participants. In cohorts 1 to 4 and 6, single doses of tarperprumig were administered via subcutaneous (SC) injection (12.5, 50, 150, 450, and 1200 mg doses). In cohorts 8 and 9, the 150 mg dose was given via SC injection once weekly for 5 doses. In cohort 5, a single dose of 450 mg was administered via intravenous infusion. Sixty participants were randomized (3:1) to tarperprumig or placebo. There were no discontinuations due to adverse events (AEs) in participants receiving tarperprumig. There were no serious AEs, events of serious infection, or deaths. No N. meningitidis infections occurred. Most AEs were mild and not treatment related. Tarperprumig exposure resulted in linear dose proportionality among all but one cohort. Mean absolute bioavailability of tarperprumig was 94%. AP activity decreased rapidly after tarperprumig administration. Complete AP inhibition (< 1% of baseline value) was observed in all cohorts except for cohort 1 (12.5 mg SC). There was no change in complement classical or lectin pathway activity. Antidrug antibody titers were mostly low and did not impact PK. Tarperprumig was well tolerated and completely inhibited the AP in healthy adults. These results support continued investigation of tarperprumig to treat diseases involving complement activation. Trial Registration: EudraCT: 2021–002472‐39/NCT04631562

Properdin, a positive regulator of complement alternative pathway, participates in renal ischemia–reperfusion (IR) injury and also acts as a pattern-recognition molecule affecting apoptotic T-cell clearance. However, the role of properdin in tubular epithelial cells (TECs) at the repair phase post IR injury is not well defined. This study revealed that properdin knockout (P KO ) mice exhibited greater injury in renal function and histology than wild-type (WT) mice post 72-h IR, with more apoptotic cells and macrophages in tubular lumina, increased active caspase-3 and HMGB1, but better histological structure at 24 h. Raised erythropoietin receptor by IR was furthered by P KO and positively correlated with injury and repair markers. Properdin in WT kidneys was also upregulated by IR, while H 2 O 2 -increased properdin in TECs was reduced by its small-interfering RNA (siRNA), with raised HMGB1 and apoptosis. Moreover, the phagocytic ability of WT TECs, analyzed by pHrodo Escherichia coli bioparticles, was promoted by H 2 O 2 but inhibited by P KO . These results were confirmed by counting phagocytosed H 2 O 2 -induced apoptotic TECs by in situ end labeling fragmented DNAs but not affected by additional serum with/without properdin. Taken together, P KO results in impaired phagocytosis at the repair phase post renal IR injury. Properdin locally produced by TECs plays crucial roles in optimizing damaged cells and regulating phagocytic ability of TECs to effectively clear apoptotic cells and reduce inflammation.

Many biopharmaceutical products exhibit extensive structural micro-heterogeneity due to an array of co-occurring post-translational modifications. These modifications often effect the functionality of the product and therefore need to be characterized in detail. Here, we present an integrative approach, combining two advanced mass spectrometry-based methods, high-resolution native mass spectrometry and middle-down proteomics, to analyse this micro-heterogeneity. Taking human erythropoietin and the human plasma properdin as model systems, we demonstrate that this strategy bridges the gap between peptide- and protein-based mass spectrometry platforms, providing the most complete profiling of glycoproteins. Integration of the two methods enabled the discovery of three undescribed C -glycosylation sites on properdin, and revealed in addition unexpected heterogeneity in occupancies of C -mannosylation. Furthermore, using various sources of erythropoietin we define and demonstrate the usage of a biosimilarity score to quantitatively assess structural similarity, which would also be beneficial for profiling other therapeutic proteins and even plasma protein biomarkers. Many biopharmaceuticals exhibit mixed heterogeneity in their post-translational modifications (PTMs) that are essential for their function. Here the authors use a combination of mass spectrometry techniques to analyse human erythropoietin (EPO) and properdin to discover new PTMs on properdin and derive a biosimilarity score for various sources of EPO.

Properdin and factor H (FH), the two regulatory proteins of the alternative complement pathway, oppose each other to maintain the complement system's activation. While properdin upregulates, FH downregulates the complement alternative pathway. The current study evaluated the expression of properdin and FH transcripts and proteins in the placental tissues and umbilical cords (UC) of preeclampsia (PE), gestational diabetes mellitus (GDM), and recurrent pregnancy loss (RPL) compared to normal healthy pregnancy (N). The tissue histology of PE, GDM and RPL were observed using haematoxylin-eosin and Masson's trichrome staining. To understand the expression and distribution of properdin and FH, RT-qPCR, western blot, and immunohistochemistry were carried out. The expressions of two additional complement components, C3 and C5, were also detected by western blot. The placentae from PE and GDM showed substantial collagen and fibrinoid deposition, thicker foetal blood capillaries, and a considerable number of syncytial knots. There was a significant rise in the level of properdin and significant decline in the level of FH at both mRNA and protein levels in the placentae and umbilical cord of PE compared to N; in GDM placentae, both properdin and FH were significantly elevated compared to N. In the case of RPL placentae, similar to PE, properdin expression was high while FH expression level was low. In both PE and RPL placentae, C3 and C5 levels were high, suggesting possibility of overactivation of complement proteins in the placenta. The observed elevated properdin level can contribute to the heightened inflammatory response in PE, GDM and RPL placentae. Low FH and high C3 and C5 in the placenta possibly suggests dysregulated complement activation in PE and RPL.

The alternative pathway (AP) of complement activation is constitutively active and must be regulated by host proteins to prevent autologous tissue injury. Dysfunction of AP regulatory proteins has been linked to several human inflammatory disorders. Properdin is a positive regulator of AP complement activation that has been shown to extend the half-life of cell surface–bound C3 convertase C3bBb; it may also initiate AP complement activation. Here, we demonstrate a critical role for properdin in autologous tissue injury mediated by AP complement activation. We identified myeloid lineage cells as the principal source of plasma properdin by generating mice with global and tissue-specific knockout of Cfp (which encodes properdin) and by generating BM chimeric mice. Properdin deficiency rescued mice from AP complement–mediated embryonic lethality caused by deficiency of the membrane complement regulator Crry and markedly reduced disease severity in the K/BxN model of arthritis. Ab neutralization of properdin in WT mice similarly ameliorated arthritis development, whereas reconstitution of properdin-null mice with exogenous properdin restored arthritis sensitivity. These data implicate systemic properdin as a key contributor to AP complement–mediated injury and support its therapeutic targeting in complement-dependent human diseases.

Emerging urinary biomarkers continue to show promise in evaluating lupus nephritis (LN). Here, we screen urine from active LN patients for 1129 proteins using an aptamer-based platform, followed by ELISA validation in two independent cohorts comprised of 127 inactive lupus, 107 active LN, 67 active non-renal lupus patients and 74 healthy controls, of three different ethnicities. Urine proteins that best distinguish active LN from inactive disease are ALCAM, PF-4, properdin, and VCAM-1 among African-Americans, sE-selectin, VCAM-1, BFL-1 and Hemopexin among Caucasians, and ALCAM, VCAM-1, TFPI and PF-4 among Asians. Most of these correlate significantly with disease activity indices in the respective ethnic groups, and surpass conventional metrics in identifying active LN, with better sensitivity, and negative/positive predictive values. Several elevated urinary molecules are also expressed within the kidneys in LN, based on single-cell RNAseq analysis. Longitudinal studies are warranted to assess the utility of these biomarkers in tracking lupus nephritis. Developing noninvasive diagnostic biomarkers for lupus nephritis (LN) diagnosis is an important clinical goal. Here the authors identify urinary proteins correlated with active LN and disease severity, which differ across ethnicities but collectively outperform the current clinical method.

The complement system, an important arm of the innate immune system, is activated in heart failure (HF). We hypothesized that HF patients are characterized by an imbalance of alternative amplification loop components; including properdin and complement factor D and the alternative pathway inhibitor factor H. These components and the activation product, terminal complement complex (TCC), were measured in plasma from 188 HF patients and 67 age- and sex- matched healthy controls by enzyme immunoassay. Our main findings were: (i) Compared to controls, patients with HF had significantly increased levels of factor D and TCC, and decreased levels of properdin, particularly patients with advanced clinical disorder (i.e., NYHA functional class IV), (ii) Levels of factor D and properdin in HF patients were correlated with measures of systemic inflammation (i.e., C-reactive protein), neurohormonal deterioration (i.e., Nt-proBNP), cardiac function, and deteriorated diastolic function, (iii) Low levels of factor H and properdin were associated with adverse outcome in univariate analysis and for factor H, this was also seen in an adjusted model. Our results indicate that dysregulation of circulating components of the alternative pathway explain the increased degree of complement activation and is related to disease severity in HF patients.