C4b-binding Protein Research Articles

PepO and CppA modulate Streptococcus sanguinis susceptibility to complement immunity and virulence

ABSTRACT Streptococcus sanguinis is a ubiquitous commensal species of the oral cavity commonly involved as an opportunistic pathogen in cardiovascular infections. In this study, we investigated the functions of endopeptidase O (PepO) and a C3-degrading protease (CppA) in the systemic virulence of S. sanguinis. Isogenic mutants of pepO and cppA obtained in strain SK36 showed increased susceptibility to C3b deposition and to opsonophagocytosis by human polymorphonuclear neutrophils (PMN). These mutants differ, however, in their profiles of binding to serum amyloid P component (SAP) and C1q, whereas both showed reduced interaction with C4b-binding protein (C4BP) and/or factor H (FH) regulators as compared to SK36. The two mutants showed defects in ex vivo persistence in human blood, serum-mediated invasion of HCAEC endothelial cells, and virulence in a Galleria mellonella infection model. The transcriptional activities of pepO and cppA, assessed by RT-qPCR in nine wild-type strains, further indicated strain-specific profiles of pepO/cppA expression. Moreover, non-conserved amino acid substitutions were detected among the strains, mostly in CppA. Phylogenetic comparisons with homologues of streptococcal species of the oral and oropharyngeal sites suggested that S. sanguinis PepO and CppA have independent ancestralities. Thus, this study showed that PepO and CppA are complement evasion proteins expressed by S. sanguinis in a strain-specific manner, which are required for multiple functions associated with cardiovascular virulence.

A conserved 3D pattern in a Streptococcus pyogenes M protein immunogen elicits M-type crossreactivity

Coiled coil-forming M proteins of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes (strep A) are immunodominant targets of opsonizing antibodies. However, antigenic sequence variability of M proteins into >220M types, as defined by their hypervariable regions (HVRs), is considered to limit M proteins as vaccine immunogens because of type specificity in the antibody response. Surprisingly, a multi-HVR immunogen in clinical vaccine trials was shown to elicit M-type crossreactivity. The basis for this crossreactivity is unknown but may be due in part to antibody recognition of a 3D pattern conserved in many M protein HVRs that confers binding to human complement C4b-binding protein (C4BP). To test this hypothesis, we investigated whether a single M protein immunogen carrying the 3D pattern would elicit crossreactivity against other M types carrying the 3D pattern. We found that a 34-amino acid sequence of S.pyogenes M2 protein bearing the 3D pattern retained full C4BP-binding capacity when fused to a coiled coil-stabilizing sequence from the protein GCN4. We show that this immunogen, called M2G, elicited cross-reactive antibodies against a number of M types that carry the 3D pattern but not against those that lack the 3D pattern. We further show that the M2G antiserum-recognized M proteins displayed natively on the strep A surface and promoted the opsonophagocytic killing of strep A strains expressing these M proteins. As C4BP binding is a conserved virulence trait of strep A, we propose that targeting the 3D pattern may prove advantageous in vaccine design.

Complement Activation-Independent Attenuation of SARS-CoV-2 Infection by C1q and C4b-Binding Protein.

The complement system is a key component of the innate immune response to viruses and proinflammatory events. Exaggerated complement activation has been attributed to the induction of a cytokine storm in severe SARS-CoV-2 infection. However, there is also an argument for the protective role of complement proteins, given their local synthesis or activation at the site of viral infection. This study investigated the complement activation-independent role of C1q and C4b-binding protein (C4BP) against SARS-CoV-2 infection. The interactions of C1q, its recombinant globular heads, and C4BP with the SARS-CoV-2 spike and receptor binding domain (RBD) were examined using direct ELISA. In addition, RT-qPCR was used to evaluate the modulatory effect of these complement proteins on the SARS-CoV-2-mediated immune response. Cell binding and luciferase-based viral entry assays were utilised to assess the effects of C1q, its recombinant globular heads, and C4BP on SARS-CoV-2 cell entry. C1q and C4BP bound directly to SARS-CoV-2 pseudotype particles via the RBD domain of the spike protein. C1q via its globular heads and C4BP were found to reduce binding as well as viral transduction of SARS-CoV-2 spike protein expressing lentiviral pseudotypes into transfected A549 cells expressing human ACE2 and TMPRSS2. Furthermore, the treatment of the SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes with C1q, its recombinant globular heads, or C4BP triggered a reduction in mRNA levels of proinflammatory cytokines and chemokines such as IL-1β, IL-8, IL-6, TNF-α, IFN-α, and RANTES (as well as NF-κB) in A549 cells expressing human ACE2 and TMPRSS2. In addition, C1q and C4BP treatment also reduced SARS-CoV-2 pseudotype infection-mediated NF-κB activation in A549 cells expressing human ACE2 and TMPRSS2. C1q and C4BP are synthesised primarily by hepatocytes; however, they are also produced by macrophages, and alveolar type II cells, respectively, locally at the pulmonary site. These findings support the notion that the locally produced C1q and C4BP can be protective against SARS-CoV-2 infection in a complement activation-independent manner, offering immune resistance by inhibiting virus binding to target host cells and attenuating the infection-associated inflammatory response.

C4b-binding protein inhibits particulate- and crystalline-induced NLRP3 inflammasome activation.

Dysregulated NLRP3 inflammasome activation drives a wide variety of diseases, while endogenous inhibition of this pathway is poorly characterised. The serum protein C4b-binding protein (C4BP) is a well-established inhibitor of complement with emerging functions as an endogenously expressed inhibitor of the NLRP3 inflammasome signalling pathway. Here, we identified that C4BP purified from human plasma is an inhibitor of crystalline- (monosodium urate, MSU) and particulate-induced (silica) NLRP3 inflammasome activation. Using a C4BP mutant panel, we identified that C4BP bound these particles via specific protein domains located on the C4BP α-chain. Plasma-purified C4BP was internalised into MSU- or silica-stimulated human primary macrophages, and inhibited MSU- or silica-induced inflammasome complex assembly and IL-1β cytokine secretion. While internalised C4BP in MSU or silica-stimulated human macrophages was in close proximity to the inflammasome adaptor protein ASC, C4BP had no direct effect on ASC polymerisation in in vitro assays. C4BP was also protective against MSU- and silica-induced lysosomal membrane damage. We further provide evidence for an anti-inflammatory function for C4BP in vivo, as C4bp-/- mice showed an elevated pro-inflammatory state following intraperitoneal delivery of MSU. Therefore, internalised C4BP is an inhibitor of crystal- or particle-induced inflammasome responses in human primary macrophages, while murine C4BP protects against an enhanced inflammatory state in vivo. Our data suggests C4BP has important functions in retaining tissue homeostasis in both human and mice as an endogenous serum inhibitor of particulate-stimulated inflammasome activation.

Targeted Blood Plasma Proteomics and Hemostasis Assessment of Post COVID-19 Patients with Acute Myocardial Infarction

The molecular mechanisms underlying cardiovascular complications after the SARS-CoV-2 infection remain unknown. The goal of our study was to analyze the features of blood coagulation, platelet aggregation, and plasma proteomics in COVID-19 convalescents with AMI. The study included 66 AMI patients and 58 healthy volunteers. The groups were divided according to the anti-N IgG levels (AMI post-COVID (n = 44), AMI control (n = 22), control post-COVID (n = 31), and control (n = 27)). All participants underwent rotational thromboelastometry, thrombodynamics, impedance aggregometry, and blood plasma proteomics analysis. Both AMI groups of patients demonstrated higher values of clot growth rates, thrombus size and density, as well as the elevated levels of components of the complement system, proteins modifying the state of endothelium, acute-phase and procoagulant proteins. In comparison with AMI control, AMI post-COVID patients demonstrated decreased levels of proteins connected to inflammation and hemostasis (lipopolysaccharide-binding protein, C4b-binding protein alpha-chain, plasma protease C1 inhibitor, fibrinogen beta-chain, vitamin K-dependent protein S), and altered correlations between inflammation and fibrinolysis. A new finding is that AMI post-COVID patients opposite the AMI control group, are characterized by a less noticeable growth of acute-phase proteins and hemostatic markers that could be explained by prolonged immune system alteration after COVID-19.

Evasion of the complement system by Leishmania through the uptake of C4bBP, a complement regulatory protein, and probably by the action of GP63 on C4b molecules deposited on parasite surface

The complement system is a primary component of the vertebrate innate immune system, and its activity is harmful to microorganisms and parasites. To evade complement attack, some pathogens, such as viruses, bacteria, and protozoa, can interact with complement regulatory proteins from their hosts. Our research group has described the ability of Leishmania species to bind Factor H from human serum and use it as a tool to evade the complement system. However, there is no description of the interaction of Leishmania with other complement regulatory proteins, such as the C4b-binding protein (C4bBP), a negative regulator of classical and lectins complement system pathways. The results presented in this manuscript suggest that Leishmania infantum, L. amazonensis, and L. braziliensis recruit C4bBP from human serum. The uptake of C4bBP by L. infantum was studied in detail to improve our understanding of this inhibitory mechanism. When exposed to this complement regulator, parasites with inactivated GP63 bind to C4bBP and inactivate C4b deposited on their surface after serum exposure. This inactivation occurs by the action of Factor I, a complement system protease. In addition to the C4bBP-Factor I inactivation mechanism, the surface parasite protease GP63 can also inactivate soluble C4b molecules and probably that C4b molecules deposited on the parasites surface. This manuscript shows that Leishmania has two independent strategies to inactivate C4b molecules, preventing the progress of classical and lectins pathways. The identification of the C4bBP receptor on the Leishmania membrane may provide a new vaccine target to fight leishmaniasis.

Neisseria gonorrhoeae co-opts C4b-binding protein to enhance complement-independent survival from neutrophils.

Neisseria gonorrhoeae (Gc) is a human-specific pathogen that causes the sexually transmitted infection gonorrhea. Gc survives in neutrophil-rich gonorrheal secretions, and recovered bacteria predominantly express phase-variable, surface-expressed opacity-associated (Opa) proteins (Opa+). However, expression of Opa proteins like OpaD decreases Gc survival when exposed to human neutrophils ex vivo. Here, we made the unexpected observation that incubation with normal human serum, which is found in inflamed mucosal secretions, enhances survival of Opa+ Gc from primary human neutrophils. We directly linked this phenomenon to a novel complement-independent function for C4b-binding protein (C4BP). When bound to the bacteria, C4BP was necessary and sufficient to suppress Gc-induced neutrophil reactive oxygen species production and prevent neutrophil phagocytosis of Opa+ Gc. This research identifies for the first time a complement-independent role for C4BP in enhancing the survival of a pathogenic bacterium from phagocytes, thereby revealing how Gc exploits inflammatory conditions to persist at human mucosal surfaces.

A Novel Electrochemiluminescence Immunosensor Based on Resonance Energy Transfer between g-CN and NU-1000(Zr) for Ultrasensitive Detection of Ochratoxin A in Coffee.

In this study, an electrochemiluminescence (ECL) immunosensor based on nanobody heptamer and resonance energy transfer (RET) between g-C3N4 (g-CN) and NU-1000(Zr) was proposed for ultrasensitive ochratoxin A (OTA) detection. First, OTA heptamer fusion protein was prepared by fusing OTA-specific nanometric (Nb28) with a c-terminal fragment of C4 binding protein (C4bpα) (Nb28-C4bpα). Then, Nb28-C4bpα heptamer with the high affinity used as a molecular recognition probe, of which plenty of binding sites were provided for OTA-Apt-NU-1000(Zr) nanocomposites, thereby improving the immunosensors' sensitivity. In addition, the quantitative analysis of OTA can be achieved by using the signal quenching effect of NU-1000(Zr) on g-CN. As the concentration of OTA increases, the amount of OTA-Apt-NU-1000(Zr) fixed on the electrode surface decreases. RET between g-CN and NU-1000(Zr) is weakened leading to the increase of ECL signal. Thus, OTA content is indirectly proportional to ECL intensity. Based on the above principle, an ultra-sensitive and specific ECL immunosensor for OTA detection was constructed by using heptamer technology and RET between two nanomaterials, with a range from 0.1 pg/mL to 500 ng/mL, and the detection limit of only 33 fg/mL. The prepared ECL-RET immunosensor showed good performance and can be successfully used for the determination of OTA content in real coffee samples, suggesting that the nanobody polymerization strategy and the RET effect between NU-1000(Zr) and g-CN can provide an alternative for improving the sensitivity of important mycotoxin detection.

Proteomic analysis of small extracellular vesicles from the plasma of patients with hepatocellular carcinoma

PurposeLiver cancer is one of the most common tumors with the seventh-highest incidence and the third-highest mortality. Many studies have shown that small extracellular vesicles (sEVs) play an important role in liver cancer. Here, we report comprehensive signatures for sEV proteins from plasma obtained from patients with hepatocellular carcinoma (HCC), which might be valuable for the evaluation and diagnosis of HCC.MethodsWe extracted sEVs from the plasma of controls and patients with HCC. Differentially expressed proteins in the sEVs were analyzed using label-free quantification and bioinformatic analyses. Western blotting (WB) was used to validate the abovementioned sEV proteins.ResultsProteomic analysis was performed for plasma sEVs from 21 patients with HCC and 15 controls. Among the 335 identified proteins in our study, 27 were significantly dysregulated, including 13 upregulated proteins that were involved predominantly in the complement cascade (complement C1Q subcomponent subunit B (C1QB), complement C1Q subcomponent subunit C (C1QC), C4B-binding protein alpha chain (C4BPA), and C4B-binding protein beta chain (C4BPB)) and the coagulation cascade (F13B, fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), and fibrinogen gamma chain (FGG)). We verified increased levels of the C1QB, C1QC, C4BPA, and C4BPB proteins in the plasma sEVs from patients with HCC in both the discovery cohort and validation cohort.ConclusionsThe complement cascade in sEVs was significantly involved in HCC progression. C1QB, C1QC, C4BPA, and C4BPB were highly abundant in the plasma sEVs from patients with HCC and might represent molecular signatures.

Interactions of Candida tropicalis pH-related antigen 1 with complement proteins C3, C3b, factor-H, C4BP and complement evasion

Candida, as a part of the human microbiota, can cause opportunistic infections that are either localised or systemic candidiasis. Emerging resistance to the standard antifungal drugs is associated with increased mortality rate due to invasive Candida infections, particularly in immunocompromised patients. While there are several species of Candida, an increasing number of Candida tropicalis isolates have been recently reported from patients with invasive candidiasis or inflammatory bowel diseases. In order to establish infections, C. tropicalis has to adopt several strategies to escape the host immune attack. Understanding the immune evasion strategies is of great importance as these can be exploited as novel therapeutic targets. C. albicans pH-related antigen 1 (CaPra1), a surface bound and secretory protein, has been found to interact strongly with the immune system and help in complement evasion. However, the role of C. tropicalis Pra1 (CtPra1) and its interaction with the complement is not studied yet. Thus, we characterised how pH-related antigen 1 of C. tropicalis (CtPra1) interacts with some of the key complement proteins of the innate immune system. CtPra1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. Recombinant CtPra1, was found to bind human C3 and C3b, central molecules of the complement pathways that are important components of the innate immune system. It was also found to bind human complement regulatory proteins factor-H and C4b-binding protein (C4BP). CtPra1-factor-H and CtPra1-C4BP interactions were found to be ionic in nature as the binding intensity affected by high sodium chloride concentrations. CtPra1 inhibited functional complement activation with different effects on classical (∼20 %), lectin (∼25 %) and alternative (∼30 %) pathways. qPCR experiments using C. tropicalis clinical isolates (oral, blood and peritoneal fluid) revealed relatively higher levels of expression of CtPra1 gene when compared to the reference strain. Native CtPra1 was found to be expressed both as membrane-bound and secretory forms in the clinical isolates. Thus, C. tropicalis appears to be a master of immune evasion by using Pra1 protein. Further investigation using in-vivo models will help ascertain if these proteins can be novel therapeutic targets.

Hijacking the human complement inhibitor C4b-binding protein by the sporozoite stage of the Plasmodium falciparum parasite.

The complement system is considered the first line of defense against pathogens. Hijacking complement regulators from blood is a common evasion tactic of pathogens to inhibit complement activation on their surfaces. Here, we report hijacking of the complement C4b-binding protein (C4bp), the regulator of the classical and lectin pathways of complement activation, by the sporozoite (SPZ) stage of the Plasmodium falciparum parasite. This was shown by direct binding of radiolabeled purified C4bp to live SPZs as well as by binding of C4bp from human serum to SPZs in indirect immunofluorescence assays. Using a membrane-bound peptide array, peptides from the N-terminal domain (NTD) of P. falciparum circumsporozoite protein (CSP) were found to bind C4bp. Soluble biotinylated peptide covering the same region on the NTD and a recombinantly expressed NTD also bound C4bp in a dose-dependent manner. NTD-binding site on C4bp was mapped to the CCP1-2 of the C4bp α-chain, a common binding site for many pathogens. Native CSP was also co-immunoprecipitated with C4bp from human serum. Preventing C4bp binding to the SPZ surface negatively affected the SPZs gliding motility in the presence of functional complement and malaria hyperimmune IgG confirming the protective role of C4bp in controlling complement activation through the classical pathway on the SPZ surface. Incorporating the CSP-C4bp binding region into a CSP-based vaccine formulation could induce vaccine-mediated immunity that neutralizes this immune evasion region and increases the vaccine efficacy.

Skin Necrosis in a Patient with Protein S Deficiency, Warfarin Anticoagulation, and Acute COVID-19 Infection: A Case Report

Abstract Introduction/Objective Although thrombotic complications from COVID are well documented, the pathophysiology remains poorly understood, especially in patients with comorbid hypercoagulable risk factors and anticoagulant medication use. COVID has several dermatologic manifestations that may occur due to alterations in hemostasis. We present a case of skin necrosis in a patient with protein S deficiency, warfarin anticoagulation, and acute COVID infection. Methods/Case Report A 48-year-old female, BMI >50, heterozygous Protein S (PS) deficiency, Factor V Leiden, and history of recurrent venous thromboembolic events on warfarin, tested positive for COVID. Her only symptom was fatigue. Several days later, the patient developed painful ecchymoses and erythematous blisters on her breast, arm, and abdomen. Laboratory results showed INR 3.6, fibrinogen 675-1054 gm/dL, WBC 15k with absolute neutrophilia, and D-dimer 2274 ug/L DDU. The patient confirmed compliance with her maintenance warfarin dose. Biopsy showed thrombotic vasculopathy without leukocytoclastic vasculitis. Wound cultures were negative. Results (if a Case Study enter NA) NA. Conclusion The patient’s history and presentation were concerning for warfarin induced skin necrosis. However, the patient had taken warfarin for > 20 years and had an INR of 3.6 which substantiated that warfarin was not discontinued or missed. A retrospective review of INR results during acute COVID infection showed a decrease in time in therapeutic range and an increase in supratherapeutic INRs, suggesting COIVD patients may require decreased warfarin doses. Additionally, PS levels decline in patients with COVID; a similar effect is seen with HIV and Varicella infections. It is theorized that this may be mediated by an increase in C4b binding protein, an acute phase reactant that regulates the complement cascade and binds PS. With an infectious source ruled out and the patients’ known comorbid conditions, an acute exacerbation of the patients underlying hypercoagulable state is the likely driver of the patient’s skin necrosis with COVID as the probable precipitator. In summary, inherited and acquired hypercoagulable risk factors should be included in the differential diagnosis as a cause of skin changes in patients with COVID infection. Although histology may be helpful in diagnosing the cause of skin changes with COVID, laboratory results and clinical correlation are key in identifying a etiology.

Simultaneous heptamerization of nanobody and alkaline phosphatase by self-assembly and its application for ultrasensitive immunodetection of small molecular contaminants in agro-products

Antibody affinity and enzyme loads are two critical factors in determining the sensitivity of enzyme-linked immunosorbent assay for small molecular contaminants in food. Multimerization is considered an effective strategy to improve antibody affinity and enzyme loads. Herein, we selected ochratoxin A (OTA) as the model antigen and constructed a nanobody-alkaline phosphatase (Nb-ALP) heptamer fusion for OTA, where the Nb and ALP were simultaneously heptamerized via the self-associating peptide from the α-chains of C4 binding protein (C4bpα). The heptamer was used to develop an enhanced colorimetric enzyme immunoassay for OTA. Under the optimal experimental parameters, the proposed method has a half maximal inhibitory concentration of 0.081 ng/mL for OTA, which is 12.5-fold lower than the Nb-ALP-based method. Negligible cross-reaction was observed with OTA analogs and other common cereal mycotoxins. Moreover, good recovery rates and repeatability were obtained in the spiked cereal samples. The developed method was then applied to the analysis of OTA in real agro-products, and the results correlated well with that of the LC-MS/MS method. Overall, this work demonstrated that the simultaneous heptamerization of nanobody and alkaline phosphatase by self-assembly is an effective strategy for improving antibody affinity and enzyme loads. It also showed the great potential of Nb–C4bpα-ALP heptamer as a robust dual-functional probe for sensitive, selective, and rapid detection of mycotoxin and other small molecular contaminants in food. • Dual-functional nanobody-alkaline phosphatase heptamer (Nb–C4bpα-ALP) for OTA. • Simultaneous heptamerization of Nb and ALP by the self-assembly strategy. • Nb–C4bpα-ALP shows better binding affinity and enzyme activity than Nb-ALP. • Nb–C4bpα-ALP-based enzyme immunoassay for small molecules was first reported. • The developed method has an IC 50 of 0.081 ng/mL with the assay time of 20 min.

Association Between the C4 Binding Protein Level and White Matter Integrity in Major Depressive Disorder.

Objective Considerable evidence suggests that neuroinflammation plays an important role in the pathophysiology of major depressive disorder (MDD). However, the relationship between serum C4 binding protein (C4BP) and white matter (WM) tract integrity in MDD has not been investigated. Methods We obtained diffusion tensor images of 44 patients with MDD and 44 healthy controls and performed TRActs Constrained by UnderLying Anatomy (TRACULA) analysis to assess WM tract integrity. Serum C4-binding protein alpha chain (C4BPA) and C4- binding protein beta chain (C4BPB) levels were measured and in-between group comparisons were obtained. The correlation between serum C4BP levels and WM tract integrity was examined. Results In comparison to healthy controls, both serum C4BPA and C4BPB were higher in MDD. Also, fractional anisotropy (FA) was increased in the left cingulum-angular bundle (CAB) in MDD, but not healthy controls (HCs). A significant correlation was found between serum C4BP and FA levels in the right cingulum-cingulate gyrus bundle (CCG) in MDD. Conclusion This study is the first to investigate the correlation between serum C4BP levels and WM tract integrity in MDD. We identified an increase in WM integrity in the left CAB region in MDD. Furthermore, serum C4BP levels were higher in MDD, and this finding correlated with increased WM integrity in the right CCG region.

Quantitative Plasma Proteomics to Identify Candidate Biomarkers of Relapse in Pediatric/Adolescent Hodgkin Lymphoma.

Classical pediatric Hodgkin Lymphoma (HL) is a rare malignancy. Therapeutic regimens for its management may be optimized by establishing treatment response early on. The aim of this study was to identify plasma protein biomarkers enabling the prediction of relapse in pediatric/adolescent HL patients treated under the pediatric EuroNet-PHL-C2 trial. We used untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics at the time of diagnosis—before any therapy—as semiquantitative method to profile plasma proteins specifically associated with relapse in 42 children with nodular sclerosing HL. In both the exploratory and the validation cohorts, six proteins (apolipoprotein E, C4b-binding protein α chain, clusterin, fibrinogen γ chain, prothrombin, and vitronectin) were more abundant in the plasma of patients whose HL relapsed (|fold change| ≥ 1.2, p < 0.05, Student’s t-test). Predicting protein function with the Gene Ontology classification model, the proteins were included in four biological processes (p < 0.01). Using immunoblotting and Luminex assays, we validated two of these candidate biomarkers—C4b-binding protein α chain and clusterin—linked to innate immune response function (GO:0045087). This study identified C4b-binding protein α chain and clusterin as candidate early plasma biomarkers of HL relapse, and important for the purpose of shedding light on the molecular scenario associated with immune response in patients treated under the EuroNet-PHL-C2 trial.

Exosome Proteomics Reveals the Deregulation of Coagulation, Complement and Lipid Metabolism Proteins in Gestational Diabetes Mellitus.

Exosomes are small extracellular vesicles with a variable protein cargo in consonance with cell origin and pathophysiological conditions. Gestational diabetes mellitus (GDM) is characterized by different levels of chronic low-grade inflammation and vascular dysfunction; however, there are few data characterizing the serum exosomal protein cargo of GDM patients and associated signaling pathways. Eighteen pregnant women were enrolled in the study: 8 controls (CG) and 10 patients with GDM. Blood samples were collected from patients, for exosomes’ concentration. Protein abundance alterations were demonstrated by relative mass spectrometric analysis and their association with clinical parameters in GDM patients was performed using Pearson’s correlation analysis. The proteomics analysis revealed 78 significantly altered proteins when comparing GDM to CG, related to complement and coagulation cascades, platelet activation, prothrombotic factors and cholesterol metabolism. Down-regulation of Complement C3 (C3), Complement C5 (C5), C4-B (C4B), C4b-binding protein beta chain (C4BPB) and C4b-binding protein alpha chain (C4BPA), and up-regulation of C7, C9 and F12 were found in GDM. Our data indicated significant correlations between factors involved in the pathogenesis of GDM and clinical parameters that may improve the understanding of GDM pathophysiology. Data are available via ProteomeXchange with identifier PXD035673.

Total Plasma Protein S Is a Prothrombotic Marker in People Living With HIV.

HIV-1 infection is associated with multiple procoagulant changes and increased thrombotic risk. Possible mechanisms for this risk include heigthened expression of procoagulant tissue factor (TF) on circulating monocytes, extracellular vesicles, and viral particles and/or acquired deficiency of protein S (PS), a critical cofactor for the anticoagulant protein C (PC). PS deficiency occurs in up to 76% of people living with HIV-1 (PLWH). As increased ex vivo plasma thrombin generation is a strong predictor of mortality, we investigated whether PS and plasma TF are associated with plasma thrombin generation. We analyzed plasma samples from 9 healthy controls, 17 PLWH on first diagnosis (naive), and 13 PLWH on antiretroviral therapy (ART). Plasma thrombin generation, total and free PS, PC, C4b-binding protein, and TF activity were measured. We determined that the plasma thrombin generation assay is insensitive to PS, because of a lack of PC activation, and developed a modified PS-sensitive assay. Total plasma PS was reduced in 58% of the naive and 38% of the ART-treated PLWH samples and correlated with increased thrombin generation in the modified assay. Conversely, plasma TF was not increased in our patient population, suggesting that it does not significantly contribute to ex vivo plasma thrombin generation. These data suggest that reduced total plasma PS contributes to the thrombotic risk associated with HIV-1 infection and can serve as a prothrombotic biomarker. In addition, our refined thrombin generation assay offers a more sensitive tool to assess the functional consequences of acquired PS deficiency in PLWH.

CipA mediates complement resistance of Acinetobacter baumannii by formation of a factor I-dependent quadripartite assemblage.

Multidrug-resistant Acinetobacter baumannii is known to be one of the leading pathogens that cause severe nosocomial infections. To overcome eradication by the innate immune system during infection, A. baumannii developed a number of immune evasion strategies. Previously, we identified CipA as a plasminogen-binding and complement-inhibitory protein. Here we show that CipA inhibits all three complement activation pathways and interacts with key complement components C3, C3b, C4b, C5, Factor B, Factor D, and in particular Factor I. CipA also targets function of the C5 convertase as cleavage of C5 was impaired. Systematic screening of CipA variants identified two separate binding sites for C3b and a Factor I-interacting domain located at the C-terminus. Structure predictions using AlphaFold2 and binding analyses employing CipA variants lacking Factor I-binding capability confirmed that the orientation of the C-terminal domain is essential for the interaction with Factor I. Hence, our analyses point to a novel Factor I-dependent mechanisms of complement inactivation mediated by CipA of A. baumannii. Recruitment of Factor I by CipA initiates the assembly of a quadripartite complex following binding of either Factor H or C4b-binding protein to degrade C3b and C4b, respectively. Loss of Factor I binding in a CipA-deficient strain, or a strain producing a CipA variant lacking Factor I-binding capability, correlated with a higher susceptibility to human serum, indicating that recruitment of Factor I enables A. baumannii to resist complement-mediated killing.

Antithrombotic potential of a single‐domain antibody enhancing the activated protein C‐cofactor activity of protein S

BackgroundProtein S (PS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) in the proteolytic inactivation of activated factors V (FVa) and VIII (FVIIIa), but also for tissue factor pathway inhibitor α (TFPIα) in the inhibition of activated factor X (FXa). ObjectiveFor therapeutic purposes, we aimed at generating single‐domain antibodies (sdAbs) that could specifically modulate the APC‐cofactor activity of PS in vivo. MethodsA llama‐derived immune library of sdAbs was generated and screened on recombinant human PS by phage display. PS binders were tested in a global activated partial thromboplastin time (APTT)‐based APC‐cofactor activity assay. ResultsA PS‐specific sdAb (PS003) was found to enhance the APC‐cofactor activity of PS in our APTT‐based assay, and this enhancing effect was greater for a bivalent form of PS003 (PS003biv). Further characterization of PS003biv demonstrated that PS003biv also enhanced the APC‐cofactor activity of PS in a tissue factor (TF)‐induced thrombin generation assay and stimulated APC in the inactivation of FVa, but not FVIIIa, in plasma‐based assays. Furthermore, PS003biv was directed against the sex hormone‐binding globulin (SHBG)‐like domain but did not inhibit the binding of PS to C4b‐binding protein (C4BP) and did not interfere with the TFPIα‐cofactor activity of PS. In mice, PS003biv exerted an antithrombotic effect in a FeCl3‐induced thrombosis model, while not affecting physiological hemostasis in a tail‐clip bleeding model. DiscussionAltogether, these results showed that pharmacological enhancement of the APC‐cofactor activity of PS through an original anti‐PS sdAb might constitute a promising and safe antithrombotic strategy.

Differential RNA aptamer affinity profiling on plasma as a potential diagnostic tool for bladder cancer.

The molecular composition of blood is a signature of human health, reflected in the thousands of blood biomarkers known for human diseases. However, establishing robust disease markers is challenging due to the diversity of individual samples. New sequencing methods have simplified biomarker discovery for circulating DNA and RNA while protein profiling is still laborious and costly. To harness the power of high-throughput sequencing to profile the protein content of a biological sample, we developed a method termed APTASHAPE that uses oligonucleotide aptamers to recognize proteins in complex biofluids. We selected a large pool of 2′Fluoro protected RNA sequences to recognize proteins in human plasma and identified a set of 33 cancer-specific aptamers. Differential enrichment of these aptamers after selection against 1 μl of plasma from individual patients allowed us to differentiate between healthy controls and bladder cancer-diagnosed patients (91% accuracy) and between early non-invasive tumors and late stage tumors (83% accuracy). Affinity purification and mass spectrometry of proteins bound to the predictive aptamers showed the main target proteins to be C4b-binding protein, Complement C3, Fibrinogen, Complement factor H and IgG. The APTASHAPE method thus provides a general, automated and highly sensitive platform for discovering potential new disease biomarkers.