Complement Component C5 and TLR Molecule CD14 Mediate Heme-Induced Thromboinflammation in Human Blood.

Heme is a critical danger molecule liberated from hemeproteins in various conditions, including from hemoglobin in hemolytic diseases. Heme may cause thromboinflammatory damage by activating inflammatory and hemostatic pathways, such as complement, the TLRs, coagulation, and platelets. In this study, we explored the effect of single and dual inhibition of complement component C5 and TLR coreceptor CD14 on heme-induced thromboinflammation in an ex vivo human whole blood model. Heme induced a dose-dependent activation of complement via the alternative pathway. Single inhibition of C5 by eculizumab attenuated the release of IL-6, IL-8, TNF, MCP-1, MIP-1α, IFN-γ, LTB-4, MMP-8 and -9, and IL-1Ra with more than 60% (p < 0.05 for all) reduced the upregulation of CD11b on granulocytes and monocytes by 59 and 40%, respectively (p < 0.05), and attenuated monocytic tissue factor expression by 33% (p < 0.001). Blocking CD14 attenuated IL-6 and TNF by more than 50% (p < 0.05). In contrast to single inhibition, combined C5 and CD14 was required for a significantly attenuated prothrombin cleavage (72%, p < 0.05). Markers of thromboinflammation were also quantified in two patients admitted to the hospital with sickle cell disease (SCD) crisis. Both SCD patients had pronounced hemolysis and depleted plasma hemopexin and haptoglobin. Plasma heme and complement activation was markedly increased in one patient, a coinciding observation as demonstrated ex vivo. In conclusion, heme-induced thromboinflammation was largely attenuated by C5 inhibition alone, with a beneficial effect of adding a CD14 inhibitor to attenuate prothrombin activation. Targeting C5 has the potential to reduce thromboinflammation in SCD crisis patients.

In vitro evaluation of iron oxide nanoparticle-induced thromboinflammatory response using a combined human whole blood and endothelial cell model.

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.

Functional evaluation of complement factor I variants by immunoassays and SDS-PAGE.

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.

Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood.

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® 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.

Heme Interferes With Complement Factor I-Dependent Regulation by Enhancing Alternative Pathway Activation.

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.

Tailoring the specificity of the type C feruloyl esterase FoFaeC from Fusarium oxysporum towards methyl sinapate by rational redesign based on small molecule docking simulations.

The type C feruloyl esterase FoFaeC from Fusarium oxysporum is a newly discovered enzyme with high potential for use in the hydrolysis of lignocellulosic biomass but it shows low activity towards sinapates. In this work, small molecule docking simulations were employed in order to identify important residues for the binding of the four model methyl esters of hydroxycinnamic acids, methyl ferulate/caffeate/sinapate/p-coumarate, to the predicted structure of FoFaeC. Subsequently rational redesign was applied to the enzyme' active site in order to improve its specificity towards methyl sinapate. A double mutation (F230H/T202V) was considered to provide hydrophobic environment for stabilization of the methoxy substitution on sinapate and a larger binding pocket. Five mutant clones and the wild type were produced in Pichia pastoris and biochemically characterized. All clones showed improved activity, substrate affinity, catalytic efficiency and turnover rate compared to the wild type against methyl sinapate, with clone P13 showing a 5-fold improvement in catalytic efficiency. Although the affinity of all mutant clones was improved against the four model substrates, the catalytic efficiency and turnover rate decreased for the substrates containing a hydroxyl substitution.

Symptom improvement in lung cancer patients treated with erlotinib: quality of life analysis of the National Cancer Institute of Canada Clinical Trials Group Study BR.21.

PURPOSE: This report describes the quality of life (QOL) findings of a randomized placebo controlled study of erlotinib, an epidermal growth factor receptor inhibitor, in patients with non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: This double-blind phase III trial randomly assigned 731 patients with NSCLC who had progressed after prior chemotherapy to erlotinib 150 mg daily or placebo, with survival as the primary study outcome. QOL was assessed by European Organisation for Research and Treatment of Cancer QLQ-C30 and the lung cancer module QLQ-LC13. The primary end points for QOL analysis were time to deterioration of three common lung cancer symptoms: cough, dyspnea, and pain. RESULTS: Survival was significantly longer (hazard ratio, 0.70; P < .0001) in the erlotinib arm. Compliance with QOL was 87% at baseline and more than 70% during treatment. Patients receiving erlotinib had significantly longer median time to deterioration for all three symptoms (4.9 v 3.7 months for cough [P = .04]; 4.7 v 2.9 months for dyspnea [P = .04], and 2.8 v 1.9 months for pain [P = .03]). QOL response analyses showed that 44%, 34%, and 42% of patients receiving erlotinib had improvement in these three symptoms, respectively. This was accompanied by a significant improvement in the physical function (31% erlotinib v 19% placebo, P = .01), and global QOL (35% v 26%, P < .0001). Patients with complete or partial response were more likely to have improvement in the QOL response than patients with stable or progressive disease (P < .01). CONCLUSION: Erlotinib not only improves survival in previously treated patients with NSCLC, but also improves tumor-related symptoms and important aspects of QOL.