Platelet activation and the complement system are mutually dependent. Here, we investigated the effects of storage time on complement activation and platelet function in routinely produced platelet concentrates. The platelet concentrates (n = 10) were stored at 22 degrees C for seven days and assessed daily for complement and platelet activation markers. Additionally, platelet function was analyzed in terms of their responsiveness to protease-activated receptor-1 (PAR-1) and thromboxane A2 receptor (TXA(2)R) activation and their capacity to adhere to collagen. Complement activation increased over the storage period for all analyzed markers, including the C1rs/C1-INH complex (fold change (FC) = 1.9; p < 0.001), MASP-1/C1-INH complex (FC = 2.0; p < 0.001), C4c (FC = 1.8, p < 0.001), C3bc (FC = 4.0; p < 0.01), and soluble C5b-9 (FC = 1.7, p < 0.001). Furthermore, the levels of soluble platelet activation markers increased in the concentrates over the seven-day period, including neutrophil-activating peptide-2 (FC = 2.5; p < 0.0001), transforming growth factor beta 1 (FC = 1.9; p < 0.001) and platelet factor 4 (FC = 2.1; p < 0.0001). The ability of platelets to respond to activation, as measured by surface expression of CD62P and CD63, decreased by 19% and 24% (p < 0.05) for PAR-1 and 69-72% (p < 0.05) for TXA(2)R activation, respectively, on Day 7 compared to Day 1. The extent of platelet binding to collagen was not significantly impaired during storage. In conclusion, we demonstrated that complement activation increased during the storage of platelets, and this correlated with increased platelet activation and a reduced ability of the platelets to respond to, primarily, TXA(2)R activation.

The host is protected by pattern recognition systems, including complement and TLRs, which are closely cross-talking. If improperly activated, these systems might induce tissue damage and disease. Inhibition of single downstream proinflammatory cytokines, such as TNF, IL-1β, and IL-6, have failed in clinical sepsis trials, which might not be unexpected, given the substantial amounts of mediators involved in the pathogenesis of this condition. Instead, we have put forward a hypothesis of inhibition at the recognition phase by "dual blockade" of bottleneck molecules of complement and TLRs. By acting upstream and broadly, the dual blockade could be beneficial in conditions with improper or uncontrolled innate immune activation threatening the host. Key bottleneck molecules in these systems that could be targets for inhibition are the central complement molecules C3 and C5 and the important CD14 molecule, which is a coreceptor for several TLRs, including TLR4 and TLR2. This review summarizes current knowledge of inhibition of complement and TLRs alone and in combination, in both sterile and nonsterile inflammatory processes, where activation of these systems is of crucial importance for tissue damage and disease. Thus, dual blockade might provide a general, broad-acting therapeutic regimen against a number of diseases where innate immunity is improperly activated.

The impact of complement activation and its possible relation to cytokine responses during malaria pathology was investigated in plasma samples from patients with confirmed Plasmodium falciparum malaria and in human whole-blood specimens stimulated with malaria-relevant agents ex vivo. Complement was significantly activated in the malaria cohort, compared with healthy controls, and was positively correlated with disease severity and with certain cytokines, in particular interleukin 8 (IL-8)/CXCL8. This was confirmed in ex vivo-stimulated blood specimens, in which complement inhibition significantly reduced IL-8/CXCL8 release. P. falciparum malaria is associated with systemic complement activation and complement-dependent release of inflammatory cytokines, of which IL-8/CXCL8 is particularly prominent.

Background: The complement system is an essential component of our innate defense and plays a vital role in the pathogenesis of many diseases. Assessment of complement activation is critical in monitoring both disease progression and response to therapy. Complement analysis requires accurate and standardized sampling and assay procedures, which has proven to be challenging. Objective: We performed a systematic analysis of the current methods used to assess complement components and reviewed whether the identified studies performed their complement measurements according to the recommended practice regarding pre-analytical sample handling and assay technique. Results are supplemented with own data regarding the assessment of key complement biomarkers to illustrate the importance of accurate sampling and measuring of complement components. Methods: A literature search using the Pubmed/MEDLINE database was performed focusing on studies measuring the key complement components C3, C5 and/or their split products and/or the soluble variant of the terminal C5b-9 complement complex (sTCC) in human blood samples that were published between February 2017 and February 2022. The identified studies were reviewed whether they had used the correct sample type and techniques for their analyses. Results: A total of 92 out of 376 studies were selected for full-text analysis. Forty-five studies (49%) were identified as using the correct sample type and techniques for their complement analyses, while 25 studies (27%) did not use the correct sample type or technique. For 22 studies (24%), it was not specified which sample type was used. Conclusion: A substantial part of the reviewed studies did not use the appropriate sample type for assessing complement activation or did not mention which sample type was used. This deviation from the standardized procedure can lead to misinterpretation of complement biomarker levels and hampers proper comparison of complement measurements between studies. Therefore, this study underlines the necessity of general guidelines for accurate and standardized complement analysis

Systemic inflammatory conditions are often associated with hypothermia or hyperthermia. Therapeutic hypothermia is used in post-cardiac arrest and some other acute diseases. There is a need for more knowledge concerning the effect of various temperatures on the acute inflammatory response. The complement system plays a crucial role in initiating the inflammatory response. We hypothesized that temperatures above and below the physiologic 37 & DEG;C affect complement activation and cytokine production ex vivo. Lepirudin-anticoagulated human whole blood from 10 healthy donors was incubated in the presence or absence of Escherichia coli at different temperatures (4 & DEG;C, 12 & DEG;C, 20 & DEG;C, 33 & DEG;C, 37 & DEG;C, 39 & DEG;C, and 41 & DEG;C). Complement activation was assessed by the terminal C5b-9 complement complex (TCC) and the alternative convertase C3bBbP using ELISA. Cytokines were measured using a 27-plex assay. Granulocyte and monocyte activation was evaluated by CD11b surface expression using flow cytometry. A consistent increase in complement activation was observed with rising temperature, reaching a maximum at 41 & DEG;C, both in the absence (C3bBbP p < 0.05) and presence (C3bBbP p < 0.05 and TCC p < 0.05) of E. coli. Temperature alone did not affect cytokine production, whereas incubation with E. coli significantly increased cytokine levels of IL-18, IL-2, IL-6, IL-8, IFN-& gamma;, and TNF at temperatures > 20 & DEG;C. Maximum increase occurred at 39 & DEG;C. However, a consistent decrease was observed at 41 & DEG;C, significant for IL-18 (p = 0.003). Granulocyte CD11b displayed the same temperature-dependent pattern as cytokines, with a corresponding increase in endothelial cell apoptosis and necrosis. Thus, blood temperature differentially determines the degree of complement activation and cytokine release.

Bacterial and mitochondrial DNA, sharing an evolutionary origin, act as danger-associated molecular patterns in infectious and sterile inflammation. They both contain immunomodulatory CpG motifs. Interactions between CpG motifs and the complement system are sparsely described, and mechanisms of complement activation by CpG remain unclear. Lepirudin-anticoagulated human whole blood and plasma were incubated with increasing concentrations of three classes of synthetic CpGs: CpG-A, -B, and -C oligodeoxynucleotides and their GpC sequence controls. Complement activation products were analyzed by immunoassays. Cytokine levels were determined via 27-plex beads-based immunoassay, and CpG interactions with individual complement proteins were evaluated using magnetic beads coated with CpG-B. In whole blood and plasma, CpG-B and CpG-C (p < 0.05 for both), but not CpG-A (p > 0.8 for all), led to time- and dose-dependent increase of soluble C5b-9, the alternative complement convertase C3bBbP, and the C3 cleavage product C3bc. GpC-A, -B, and -C changed soluble fluid-phase C5b-9, C3bBbP, and C3bc to the same extent as CpG-A, -B, and -C, indicating a DNA backbone-dependent effect. Dose-dependent CpG-B binding was found to C1q (r = 0.83; p 5 0.006) and factor H (r = 0.93; p < 0.001). The stimulatory complement effect was partly preserved in C2-deficient plasma and completely preserved in MASP-2-deficient serum. CpG-B increased levels of IL-1 beta, IL-2, IL-6, IL-8, MCP-1, and TNF in whole blood, which were completely abolished by inhibition of C5 and C5aR1 (p < 0.05 for all). In conclusion, synthetic analogs of bacterial and mitochondrial DNA activate the complement system via the DNA backbone. We suggest that CpG-B interacts directly with classical and alternative pathway components, resulting in complement-C5aR1-dependent cytokine release.

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3(-/-) and C5(-/-) as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3(-/-): -25%, p=0.02; C5(-/-): -20% p=0.052) and newly formed bone (C3(-/-): -38%, p=0.01; C5(-/-): -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3(-/-) mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.

Inappropriate complement activation is often responsible for incompatibility reactions that occur when biomaterials are used. Complement activation is therefore a criterion included in legislation regarding biomaterials testing. However, no consensus is yet available regarding appropriate complement-activation-related test parameters. We examined protein adsorption in plasma and complement activation/cytokine release in whole blood incubated with well-characterized polymers. Strong correlations were found between the ratio of C4 to its inhibitor C4BP and generation of 10 (mainly pro-inflammatory) cytokines, including IL-17, IFN-gamma, and IL-6. The levels of complement activation products correlated weakly (C3a) or not at all (C5a, sC5b-9), confirming their poor predictive values. We have demonstrated a direct correlation between downstream biological effects and the proteins initially adhering to an artificial surface after contact with blood. Consequently, we propose the C4/C4BP ratio as a robust, predictor of biocompatibility with superior specificity and sensitivity over the current gold standard. (C) 2014 Elsevier Ltd. All rights reserved.

When a biomaterial surface comes in contact with blood an immediate adsorption of plasma proteins to the surface will occur, and the cascade systems in the blood, such as the complement, coagulation and contact system, will be activated to various degrees. The intensity of this reaction will determine the hemocompatibility of the materials. Here we present an evaluation of the link between the composition, the physico-chemical properties and the protein adsorption properties of six newly synthesized polymers (P1-P6) and the hemocompatibility.The hemocompatibility of the polymeric surfaces was evaluated in human blood plasma and whole blood. Commercially available polyvinylchloride (PVC) was used as reference material. The hemocompatibility of the polymeric surfaces was evaluated with regard to complement activation (C3a and sC5-9 generation) and coagulation activation (platelet loss and TAT-formation) and cytokine productions (27 analytes in multiplex assay) after contact with whole blood. Contact activation was quantified by analyses of FXIIa-C1INH, FXIa-C1INH, and kallikrein-C1INH complexes.Polymers P2 (p<0.05 for C3a), P3, P5 and P6 showed less complement activation, and polymers P1 and P4 (p<0.05 for platelet loss), as well as P5 and P6 showed less coagulation activation compared with reference PVC. Polymers P1-P3 induced activation of the contact system, P3 being the most potent. Secretion of 17 cytokines including chemokines and growth factors were differentially influenced by the polymers, P1 and P3 being significantly (p<0.05) more compatible for five of the analytes.Collectively these data demonstrate that the composition of the polymers clearly leads to different biological properties as a consequence of distinctive physico-chemical properties and protein adsorption patterns.1

Oxidative stress plays a vital role for the adaptive responses to physical training. However, excessive oxidative stress can precipitate cellular damage, necessitating protective mechanisms to mitigate this effect. Glucosinolates, found predominantly in cruciferous vegetables, can be converted into isothiocyanates, known for their antioxidative properties. These compounds activate crucial antioxidant defence pathways and support mitochondrial function and protein integrity under oxidative stress, in both Nrf2-dependent and independent manners. We here administered glucosinolate-rich broccoli sprouts (GRS), in a randomized double-blinded cross-over fashion to 9 healthy subjects in combination with daily intense exercise training for 7 days. We found that exercise in combination with GRS significantly decreased the levels of carbonylated proteins in skeletal muscle and the release of myeloperoxidase into blood. Moreover, it lowered lactate accumulation during submaximal exercise, and attenuated the severe nocturnal hypoglycaemic episodes seen during the placebo condition. Furthermore, GRS in combination with exercise improved physical performance, which was unchanged in the placebo condition.

Laparoscopic and open liver resection have not been compared in randomized trials. The aim of the current study was to compare the inflammatory response after laparoscopic and open resection of colorectal liver metastases (CLM) in a randomized controlled trial.This was a predefined exploratory substudy within the Oslo CoMet-study. Forty-five patients with CLM were randomized to laparoscopic (n = 23) or open (n = 22) resection. Ethylenediaminetetraacetic acid-plasma samples were collected preoperatively and at defined time points during and after surgery and snap frozen at -80 C. A total of 25 markers were examined using luminex and enzyme-linked immunosorbent assay techniques: high-mobility box group 1(HMGB-1), cell-free DNA (cfDNA), cytokines, and terminal C5b-9 complement complex complement activation.Eight inflammatory markers increased significantly from baseline: HMGB-1, cfDNA, interleukin (IL)-6, C-reactive protein, macrophage inflammatory protein -1β, monocyte chemotactic protein -1, IL-10, and terminal C5b-9 complement complex. Peak levels were reached at the end of or shortly after surgery. Five markers, HMGB-1, cfDNA, IL-6, C-reactive protein, and macrophage inflammatory protein -1β, showed significantly higher levels in the open surgery group compared with the laparoscopic surgery group.Laparoscopic resection of CLM reduced the inflammatory response compared with open resection. The lower level of HMGB-1 is interesting because of the known association with oncogenesis.

Factor I (FI) is an essential regulator of the complement system. Together with co-factors, FI degrades C3b, which inhibits further complement activation. Genetic mutations in FI are associated with pathological conditions like age-related macular degeneration and atypical hemolytic uremic syndome. Here, we evaluated eight recombinant FI genetic variants found in patients. We assessed FI's co-factor activity in the presence of two co-factors; Factor H and soluble CR1. Different analytical assays were employed; SDS-PAGE to evaluate the degradation of C3b, ELISA to measure the generation of fluid phase iC3b and the degradation of surface-bound C3b using a novel Luminex bead-based assay. We demonstrate that mutations in the FIMAC and SP domains of FI led to significantly reduced protease activity, whereas the two analyzed mutations in the LDLRA2 domain did not result in any profound changes in FI's function. The different assays employed displayed a strong positive correlation, but differences in the activity of the genetic variants Ile55Phe and Gly261Asp could only be observed by combining different methods and co-factors for evaluating FI activity. In conclusion, our results provide a new perspective regarding available diagnostic tools for assessing the impact of mutations in FI.

Iron oxide nanoparticles (IONPs) are widely used in diagnostic and therapeutic settings. Upon systemic administration, however, they are rapidly recognized by components of innate immunity, which limit their therapeutic capacity and can potentially lead to adverse side effects. IONPs were previously found to induce the inflammatory response in human whole blood, including activation of the complement system and increased secretion of cytokines. Here, we investigated the thromboinflammatory response of 10-30 nm IONPs in lepirudin anticoagulated whole blood in interplay with endothelial cells and evaluated the therapeutic effect of applying complement inhibitors to limit adverse effects related to thromboinflammation. We found that IONPs induced complement activation, primarily at the C3-level, in whole blood incubated for up to four hours at 37°C with and without human microvascular endothelial cells. Furthermore, IONPs mediated a strong thromboinflammatory response, as seen by the significantly increased release of 21 of the 27 analyzed cytokines (p<0.05). IONPs also significantly increased cell-activation markers of endothelial cells [ICAM-1 (p<0.0001), P/E-selectin (p<0.05)], monocytes, and granulocytes [CD11b (p<0.001)], and platelets [CD62P (p<0.05), CD63 (p<0.05), NAP-2 (p<0.01), PF4 (p<0.05)], and showed cytotoxic effects, as seen by increased LDH (p<0.001) and heme (p<0.0001) levels. We found that inflammation and endothelial cell activation were partly complement-dependent and inhibition of complement at the level of C3 by compstatin Cp40 significantly attenuated expression of ICAM-1 (p<0.01) and selectins (p<0.05). We show that complement activation plays an important role in the IONPs-induced thromboinflammatory response and that complement inhibition is promising in improving IONPs biocompatibility.

Hemolysis, as a result of disease or exposure to biomaterials, is characterized by excess amounts of cell-free heme intravascularly and consumption of the protective heme-scavenger proteins in plasma. The liberation of heme has been linked to the activation of inflammatory systems, including the complement system, through alternative pathway activation. Here, we investigated the impact of heme on the regulatory function of the complement system. Heme dose-dependently inhibited factor I-mediated degradation of soluble and surface-bound C3b, when incubated in plasma or buffer with complement regulatory proteins. Inhibition occurred with factor H and soluble complement receptor 1 as co-factors, and the mechanism was linked to the direct heme-interaction with factor I. The heme-scavenger protein hemopexin was the main contaminant in purified factor I preparations. This led us to identify that hemopexin formed a complex with factor I in normal human plasma. These complexes were significantly reduced during acute vasoocclusive pain crisis in patients with sickle cell disease, but the complexes were normalized at their baseline outpatient clinic visit. Hemopexin exposed a protective function of factor I activity in vitro, but only when it was present before the addition of heme. In conclusion, we present a mechanistic explanation of how heme promotes uncontrolled complement alternative pathway amplification by interfering with the regulatory capacity of factor I. Reduced levels of hemopexin and hemopexin-factor I complexes during an acute hemolytic crisis is a risk factor for heme-mediated factor I inhibition.

Background: Exposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of the complement recognition molecule C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets.Principal Findings: Human blood serum was passed over Sepharose conjugated with CS-A, and CS-A-specific binding proteins were identified by Western blotting and mass spectrometric analysis. C1q was shown to be the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and then not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were also shown to bind to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on activated platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets.

Conclusions: This study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q also seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases. 

Platelet activation during thrombotic events is closely associated with complement and contact system activation, which in turn leads to inflammation . Here we review the interactions between activated platelets and the complement and contact activation systems in clotting blood. Chondroitin sulfate A (CS-A), released from alpha granules during platelet activation, is a potent mediator of crosstalk between platelets and the complement system. CS-A activates complement in the fluid phase, generating anaphylatoxins that mediate leukocyte activation. No complement activation seems to occur on the activated platelet surface, but C3 in the form of C3(H₂O) is bound to the surfaces of activated platelets . This finding is consistent with the strong expression of membrane-bound complement regulators present at the platelet surface. CS-A exposed on the activated platelets is to a certain amount responsible for recruiting soluble regulators to the surface. Platelet-bound C3(H₂O) acts as a ligand for leukocyte CR1 (CD35), potentially enabling platelet–leukocyte interactions. In addition, platelet activation leads to the activation of contact system enzymes, which are specifically inhibited by antithrombin, rather than by C1INH, as is the case when contact activation is induced by material surfaces. Thus, in addition to their traditional role as initiators of secondary hemostasis, platelets also act as mediators and regulators of inflammation in thrombotic events.

It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfaces of nonactivated and thrombin receptor-activating peptide-activated platelets was analyzed by flow cytometry and Western blot analyses. C1q, C4, C3, and C9 were found to bind to thrombin receptor-activating peptide-activated platelets in lepirudin-anticoagulated platelet-rich plasma (PRP) and whole blood. However, inhibiting complement activation at the C1q or C3 level did not block the binding of C3 to activated platelets. Diluting PRP and chelating divalent cations also had no effect, further indicating that the deposition of complement components was independent of complement activation. Furthermore, washed, activated platelets bound added C1q and C3 to the same extent as platelets in PRP. The use of mAbs against different forms of C3 demonstrated that the bound C3 consisted of C3(H₂O). Furthermore, exogenously added soluble complement receptor 1 was shown to bind to this form of platelet-bound C3. These observations indicate that there is no complement activation on the surface of platelets under physiological conditions. This situation is in direct contrast to a number of pathological conditions in which regulators of complement activation are lacking and thrombocytopenia and thrombotic disease are the ultimate result. However, the generation of C3(H₂O) represents nonproteolytic activation of C3 and after factor I cleavage may act as a ligand for receptor binding.

Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions.

Objective: Dysregulation of the complement system has been described in patients with heart failure (HF). However, data on the alternative pathway are scarce and it is unknown if levels of factor B (FB) and the C3 convertase C3bBbP are elevated in these patients. We hypothesized that plasma levels of FB and C3bBbP would be associated with disease severity and survival in patients with HF. Methods: We analyzed plasma levels of FB, C3bBbP, and terminal C5b-9 complement complex (TCC) in 343 HF patients and 27 healthy controls. Results: Compared with controls, patients with HF had elevated levels of circulating FB (1.6-fold, p < 0.001) and C3bBbP (1.3-fold, p < 0.001). In contrast, TCC, reflecting the terminal pathway, was not significantly increased (p = 0.15 vs controls). FB was associated with NT-proBNP, troponin, eGFR, and i.e., C-reactive protein. FB, C3bBbP and TCC were not associated with mortality in HF during a mean follow up of 4.3 years. Conclusion: Our findings suggest that in patients with HF, the alternative pathway is activated. However, this is not accompanied by activation of the terminal pathway.

Objective The aetiology of thoracic aortic aneurysm (TAA) is largely unknown, but inflammation is likely to play a central role in the pathogenesis. In this present study, we aim to investigate the complement receptors in TAA. Methods Aortic tissue and blood from 31 patients with non-syndromic TAA undergoing thoracic aortic repair surgery were collected. Aortic tissue and blood from 36 patients with atherosclerosis undergoing coronary artery bypass surgery or aortic valve replacement were collected and served as control material. The expression of the complement anaphylatoxin receptors C3aR1, C5aR1 and C5aR2 in aortic tissue were examined by quantitative RT-PCR and C5aR2 protein by immunohistochemistry. Colocalisation of C5aR2 to different cell types was analysed by immunofluorescence. Complement activation products C3bc and sC5b-9 were measured in plasma. Results Compared with controls, TAA patients had substantial (73%) downregulated gene expression of C5aR2 as seen both at the mRNA (p=0.005) level and protein (p=0.03) level. In contrast, there were no differences in the expression of C3aR1 and C5aR1 between the two groups. Immunofluorescence examination showed that C5aR2 was colocalised to macrophages and T cells in the aortic media. There were no differences in the degree of systemic complement activation between the two groups. Conclusion Our findings suggest downregulation of the C5aR2, regarded to act mainly anti-inflammatory, in electively operated TAA as compared with non-aneurysmatic aortas of patients with aortic stenosis and/or coronary artery disease. This may tip the balance towards a relative increase in the inflammatory responses induced by C5aR1 and thus enhance the inflammatory processes in TAA.

Sepsis and septic shock, caused by an excessive systemic host-inflammatory response, are associated with high morbidity and mortality. The complement system and TLRs provide important pattern recognition receptors initiating the cytokine storm by extensive cross-talk. We hypothesized that double blockade of complement C5 and the TLR coreceptor CD14 could improve survival of experimental polymicrobial sepsis. Mice undergoing cecal ligation and puncture (CLP)-induced sepsis were treated with neutralizing anti-CD14 Ab biG 53, complement C5 inhibitor coversin (Ornithodoros moubata C inhibitor), or a combination thereof. The inflammatory study (24-h observation) revealed statistically significant increases in 22 of 24 measured plasma biomarkers in the untreated CLP group, comprising 14 pro- and anti-inflammatory cytokines and 8 chemokines, growth factors, and granulocyte activation markers. Single CD14 or C5 blockade significantly inhibited 20 and 19 of the 22 biomarkers, respectively. Combined CD14 and C5 inhibition significantly reduced all 22 biomarkers (mean reduction 85%; range 54-95%) compared with the untreated CLP group. Double blockade was more potent than single treatment and was required to significantly inhibit IL-6 and CXCL1. Combined inhibition significantly reduced morbidity (motility and eyelid movement) and mortality measured over 10 d. In the positive control CLP group, median survival was 36 h (range 24-48 h). Combined treatment increased median survival to 96 h (range 24-240 h) (p = 0.001), whereas survival in the single-treatment groups was not significantly increased (median and range for anti-CD14 and anti-C5 treatment were 36 h [24-48 h] and 48 h [24-96 h]). Combined with standard intervention therapy, specific blockade of CD14 and C5 might represent a promising new therapeutic strategy for treatment of polymicrobial sepsis.

To address the shortcomings associated with corneal transplants, substantial efforts have been focused on developing new modalities such as xenotransplantion. Xenogeneic corneas are anatomically and biomechanically similar to the human cornea, yet their applications require prior decellularization to remove the antigenic components to avoid rejection. In the context of bringing decellularized corneas into clinical use, sterilization is a crucial step that determines the success of the transplantation. Well-standardized sterilization methods, such as gamma irradiation (GI), have been applied to decellularized porcine corneas (DPC) to avoid graft-associated infections in human recipients. However, little is known about the effect of GI on decellularized corneal xenografts. Here, we evaluated the radiation effect on the ultrastructure, optical, mechanical and biological properties of DPC. Transmission electron microscopy revealed that gamma irradiated decellularized porcine cornea (G-DPC) preserved its structural integrity. Moreover, the radiation did not reduce the optical properties of the tissue. Neither DPC nor G-DPC led to further activation of complement system compared to native porcine cornea when exposed to plasma. Although, DPC were mechanically comparable to the native tissue, GI increased the mechanical strength, tissue hydrophobicity and resistance to enzymatic degradation. Despite these changes, human corneal epithelial, stromal, endothelial and hybrid neuroblastoma cells grew and differentiated on DPC and G-DPC. Thus, GI may achieve effective tissue sterilization without affecting critical properties that are essential for corneal transplant survival. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Inadequate supplies of donor corneas have evoked an escalating interest in corneal xenotransplantation. However, innate immune responses contribute significantly to the mechanism of xenograft rejection. We hypothesized that complement component C5 and TLR co-receptor CD14 inhibition would inhibit porcine cornea induced innate immune responses. Therefore, we measured cytokine release in human blood, induced by three forms of corneal xenografts with or without inhibitors. Native porcine cornea (NPC) induced interleukins (IL-1 beta, IL-2, IL-6, IL-8, IL-1ra), chemokines (MCP-1, MIP-1 alpha, MIP-1 beta) and other cytokines (TNF, G-CSF, INF-gamma, FGF-basic). Decellularized (DPC) and gamma-irradiated cornea (g-DPC) elevated the release of those cytokines. C5-blockade by eculizumab inhibited all the cytokines except G-CSF when induced by NPC. However, C5-blockade failed to reduce DPC and g-DPC induced cytokines. Blockade of CD14 inhibited DPC-induced cytokines except for IL-8, MCP-1, MIP-1 alpha, and G-CSF, while it inhibited all of them when induced by g-DPC. Combined blockade of C5 and CD14 inhibited the maximum number of cytokines regardless of the xenograft type. Finally, by using the TLR4 specific inhibitor Eritoran, we showed that TLR4 activation was the basis for the CD14 effect. Thus, blockade of C5, when combined with TLR4 inhibition, may have therapeutic potential in pig-to-human corneal xenotransplantation. Statement of significance Bio-engineered corneal xenografts are on the verge of becoming a viable alternative to allogenic human donor-cornea, but the host's innate immune response is still a critical barrier for graft acceptance. By overruling this barrier, limited graft availability would no longer be an issue for treating corneal diseases. We showed that the xenograft induced inflammation is initiated by the complement system and toll-like receptor activation. Intriguingly, the inflammatory response was efficiently blocked by simultaneously targeting bottleneck molecules in the complement system (C5) and the TLR co-receptor CD14 with pharmaceutical inhibitors. We postulate that a combination of C5 and CD14 inhibition could have a great therapeutic potential to overcome the immunologic barrier in pig-to-human corneal xenotransplantation. (C) 2021 The Authors. Published by Elsevier Ltd on behalf of Acta Materialia Inc.

Thrombin plays a central role in thromboinflammatory responses, but its activity is blocked in the common ex vivo human whole blood models, making an ex vivo study of thrombin effects on thromboinflammatory responses unfeasible. In this study, we exploited the anticoagulant peptide Gly-Pro-Arg-Pro (GPRP) that blocks fibrin polymerization to study the effects of thrombin on acute inflammation in response to Escherichia coli and Staphylococcus aureus. Human blood was anticoagulated with either GPRP or the thrombin inhibitor lepirudin and incubated with either E. coli or S. aureus for up to 4 h at 37 degrees C. In GPRP-anticoagulated blood, there were spontaneous elevations in thrombin levels and platelet activation, which further increased in the presence of bacteria. Complement activation and the expression of activation markers on monocytes and granulocytes increased to the same extent in both blood models in response to bacteria. Most cytokines were not elevated in response to thrombin alone, but thrombin presence substantially and heterogeneously modulated several cytokines that increased in response to bacterial incubations. Bacterial-induced releases of IL-8, MIP-1 alpha, and mip-1 beta were potentiated in the thrombin-active GPRP model, whereas the levels of IP-10, TNF, IL-6, and IL-1 beta were elevated in the thrombin-inactive lepirudin model. Complement CS-blockade, combined with CD14 inhibition, reduced the overall cytokine release significantly, both in thrombin-active and thrombin-inactive models. Our data support that thrombin itself marginally induces leukocyte-dependent cytokine release in this isolated human whole blood but is a significant modulator of bacteria-induced inflammation by a differential effect on cytokine patterns.

Introduction: Platelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy. Methods: In this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate(R) aggregometry, flow cytometry, and confocal microscopy. Results and Discussion: We found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process.

Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining regions that harbour cysteine-rich knob domains. To produce high-affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 angstrom from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors, and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.

The complex molecular and cellular biological systems that maintain host homeostasis undergo continuous crosstalk. Complement, a component of innate immunity, is one such system. Initially regarded as a system to protect the host from infection, complement has more recently been shown to have numerous other functions, including involvement in embryonic development, tissue modeling, and repair. Furthermore, the complement system plays a major role in the pathophysiology of many diseases. Through interactions with other plasma cascades, including hemostasis, complement activation leads to the broad host-protective response known as thromboinflammation. Most complement research has been limited to reductionistic models of purified components and cells and their interactions in vitro. However, to study the pathophysiology of complement-driven diseases, including the interaction between the complement system and other inflammatory systems, holistic models demonstrating only minimal interference with complement activity are needed. Here we describe two such models; whole blood anticoagulated with either the thrombin inhibitor lepirudin or the fibrin polymerization peptide blocker GPRP, both of which retain complement activity and preserve the ability of complement to be mutually reactive with other inflammatory systems. For instance, to examine the relative roles of C3 and C5 in complement activation, it is possible to compare the effects of the C3 inhibitor compstatin effects to those of inhibitors of C5 and C5aR1. We also discuss how complement is activated by both pathogen-associated molecular patterns, inducing infectious inflammation caused by organisms such as Gram-negative and Gram-positive bacteria, and by sterile damage-associated molecular patterns, including cholesterol crystals and artificial materials used in clinical medicine. When C3 is inhibited, it is important to determine the mechanism by which inflammation is attenuated, i.e., whether the attenuation derives directly from C3 activation products or via downstream activation of C5, since the mechanism involved may determine the appropriate choice of inhibitor under various conditions. With some exceptions, most inflammatory responses are dependent on C5 and C5aR1; one exception is venous air embolism, in which air bubbles enter the blood circulation and trigger a mainly C3-dependent thromboembolism, with the formation of an active C3 convertase, without a corresponding C5 activation. Under such conditions, an inhibitor of C3 is needed to attenuate the inflammation. Our holistic blood models will be useful for further studies of the inhibition of any complement target, not just C3 or C5. The focus here will be on targeting the critical complement component, activation product, or receptor that is important for the pathophysiology in a variety of disease conditions.

When an artificial biomaterial (e.g., a stent or implantable pump) is exposed to blood, plasma proteins immediately adhere to the surface, creating a new interface between the biomaterial and the blood. The recognition proteins within the complement and contact activation/coagulation cascade systems of the blood will be bound to, or inserted into, this protein film and generate different mediators that will activate polymorphonuclear leukocytes and monocytes, as well as platelets. Under clinical conditions, the ultimate outcome of these processes may be thrombotic and inflammatory reactions, and consequently the composition and conformation of the proteins in the initial layer formed on the surface will to a large extent determine the outcome of a treatment involving the biomaterial, affecting both the functionality of the material and the patient's life quality. This review presents models of biomaterial-induced activation processes and describes various strategies to attenuate potential adverse reactions by conjugating bioactive molecules to surfaces or by introducing nanostructures.

Disturbances of host integrity have the potential to evoke activation of innate immunologic and hemostatic protection mechanisms in blood. Irrespective of whether the activating stimulus is typically immunogenic or thrombotic, it will generally affect both the complement system and platelets to a certain degree. The theme of this thesis is complement and platelet activity, which is intersected in all five included papers. The initial aim was to study the responses and mechanisms of the complement cascade in relation to platelet activation. The secondary aim was to use an applied approach to regulate platelets and complement on model biomaterial and cell surfaces.   

Complement activation was found in the fluid phase in response to platelet activation in whole blood. The mechanism was traced to platelet release of stored chondroitin sulfate-A (CS-A) and classical pathway activation via C1q. C3 was detected at the platelet surface, though its binding was independent of complement activation. The inhibitors factor H and C4-binding protein (C4BP) were detected on activated platelets, and their binding was partly dependent on surface-exposed CS-A. Collectively, these results showed that platelet activation induces inflammatory complement activation in the fluid phase. CS-A was shown to be a central molecule in the complement-modulatory functions of platelets by its interaction with C1q, C4BP, and factor H.

Platelet activation and surface adherence were successfully attenuated by conjugating an ADP-degrading apyrase on a model biomaterial. Only minor complement regulation was seen, and was therefore targeted specifically on surfaces and cells by co-immobilizing a factor H-binding peptide together with the apyrase. This combined approach led to a synchronized inhibition of both platelet and complement activation at the interface of biomaterials/xenogeneic cells and blood.

In conclusion, here presents a novel crosstalk-mechanism for activation of complement when triggering platelets, which highlights the importance of regulating both complement and platelets to lower inflammatory events. In addition, a strategy to enhance the biocompatibility of biomaterials and cells by simultaneously targeting ADP-dependent platelet activation and the alternative complement C3-convertase is proposed.

Rapid and accurate serological analysis of SARS-CoV-2 antibodies is important for assessing immune protection from vaccination or infection of individuals and for projecting virus spread within a population. The quartz crystal microbalance (QCM) is a label-free flow-based sensor platform that offers an opportunity to detect the binding of a fluid-phase ligand to an immobilized target molecule in real time. A QCM-based assay was developed for the detection of SARS-CoV-2 antibody binding and evaluated for assay reproducibility. The assay was cross-compared to the Roche electrochemiluminescence assay (ECLIA) Elecsys (R) Anti-SARS-CoV-2 serology test kit and YHLO's chemiluminescence immunoassay (CLIA). The day-to-day reproducibility of the assay had a correlation of r(2) = 0.99, p < 0.001. The assay linearity was r(2) = 0.96, p < 0.001, for dilution in both serum and buffer. In the cross-comparison analysis of 119 human serum samples, 59 were positive in the Roche, 52 in the YHLO, and 48 in the QCM immunoassay. Despite differences in the detection method and antigen used for antibody capture, there was good coherence between the assays, 80-100% for positive and 96-100% for negative test results. In summation, the QCM-based SARS-CoV-2 IgG immunoassay showed high reproducibility and linearity, along with good coherence with the ELISA-based assays. Still, factors including antibody titer and antigen-binding affinity may differentially affect the various assays' responses.

Blood incompatibility reactions caused by surfaces often involve platelet activation and subsequent platelet-initiated activation of the coagulation and complement cascades. The goal of this study was to immobilize apyrase on a biomaterial surface in order to develop an enzymatically active surface that would have the capacity to inhibit platelet activation by degrading ADP. We were able to immobilize apyrase on a polystyrene surface with preservation of the enzymatic activity. We then analyzed the hemocompatibility of the apyrase surface and of control surfaces by incubation with platelet-rich plasma (PRP) or whole blood. Monitoring of markers of platelet, coagulation, and complement activation and staining of the surfaces revealed decreased levels of platelet and coagulation activation parameters on the apyrase surface. The formation of antithrombin-thrombin and antithrombin-factor XIa complexes and the extent of platelet consumption were significantly lower on the apyrase surface than on any of the control surfaces. No significant differences were seen in complement activation (C3a levels). Staining of the apyrase surface revealed low platelet adherence and no formation of granulocyte platelet complexes. These results demonstrate that it is possible to create an antithrombotic surface targeting the ADP amplification of platelet activation by immobilizing apyrase.

Thrombin activation of C5 connects thrombosis to inflammation. Complement research in whole blood ex vivo necessitates anticoagulation, which potentially interferes with the inflammatory modulation by thrombin. We challenged the concept of thrombin as an activator of native C5 by analyzing complement activation and C5 cleavage in human whole blood anticoagulated with Gly-Pro-Arg-Pro (GPRP), a peptide targeting fibrin polymerization downstream of thrombin, allowing complete endogenous thrombin generation. GPRP dose-dependently inhibited coagulation but allowed for platelet activation in accordance with thrombin generation. Spontaneous and bacterial-induced complement activation by Escherichia coli and Staphylococcus aureus, analyzed at the level of C3 and C5, were similar in blood anticoagulated with GPRP and the thrombin inhibitor lepirudin. In the GPRP model, endogenous thrombin, even at supra-physiologic concentrations, did not cleave native C5, despite efficiently cleaving commercially sourced purified C5 protein, both in buffer and when added to C5-deficient serum. In normal serum, only exogenously added, commercially sourced C5 was cleaved, whereas the native plasma C5 remained intact. Crucially, affinity-purified C5, eluted under mild conditions using an MgCl2 solution, was not cleaved by thrombin. Acidification of plasma to pH # 6.8 by hydrochloric or lactic acid induced a C5 antigenic change, nonreversible by pH neutralization, that permitted cleavage by thrombin. Circular dichroism on purified C5 confirmed the structural change during acidification. Thus, we propose that pH-induced conformational change allows thrombin-mediated cleavage of C5 and that, contrary to previous reports, thrombin does not cleave plasma C5 in its native form, suggesting that thrombin cleavage of C5 may be restricted to certain pathophysiological conditions.

Activation of the thrombotic and complement systems is the main recognition and effector mechanisms in the multiple adverse biological responses triggered when biomaterials or therapeutic cells come into blood contact. We have created a surface which is auto-protective to human innate immunity by combining three fundamentally different strategies, all developed by us previously, which have been shown to induce substantial, but incomplete hemocompatibility when used separately. In summary, we have conjugated a factor H-binding peptide; and an ADP-degrading enzyme; using a PEG linker on both material and cellular surfaces. When exposed to human whole blood, factor H was specifically recruited to the modified surfaces and inhibited complement attack. In addition, activation of platelets and coagulation was efficiently attenuated, by degrading ADP. Thus, by inhibiting thromboinflammation using a multicomponent approach, we have created a hybrid surface with the potential to greatly reduce incompatibility reactions involving biomaterials and transplantation.

Enzyme-linked immunosorbent assay (ELISA) enables fast and simple quantification of analytes in the pico- to nanogram range in complex samples. Here, we describe an ELISA for the detection of porcine C3a as a marker for complement activation. Antibody specificity is critical for a robust assay. This assay is based on a pair of antibodies specific for the porcine C3a molecule and thus does not react with native C3.