Background Animal and observational studies have suggested a pathophysiological role for complement in venous thromboembolism (VTE), but the initiating mechanisms are unknown. Mannose‐binding lectin (MBL) bound to altered host cells leads to activation of the lectin complement pathway, and both high and low MBL levels have been implicated in the pathophysiology of cardiovascular disease. Objectives To investigate the association between plasma MBL levels and future risk of incident VTE. Methods We conducted a nested case‐control study in 417 VTE patients and 849 age‐matched and sex‐matched controls derived from the general population (Tromsø Study). Plasma MBL levels were measured using enzyme‐linked immunosorbent assay. Logistic regression models were used to estimate odds ratio (OR) for VTE across quartiles of plasma MBL levels. Results Subjects with plasma MBL levels in the lowest quartile (<435 ng/mL) had a reduced OR for overall VTE (OR 0.79, 95% confidence interval [CI]: 0.56‐1.10) and for DVT (OR 0.70, 95% CI: 0.47‐1.04) compared to those with MBL in the highest quartile (≥2423 ng/mL) after multivariable adjustments. For VTE, DVT, and pulmonary embolism (PE) the ORs decreased substantially with decreasing time between blood sampling and VTE event. Conclusions Our findings suggest that low plasma MBL levels are associated with reduced risk of VTE, and DVT in particular.

Background: It remains uncertain whether activation of the complement system, assessed by the soluble terminal C5b-9 complement complex (plasma TCC), is associated with future risk of incident venous thromboembolism (VTE). Objectives:To investigate the association between plasma levels of TCC and future risk of incident VTE in a nested case-control study, and to explore genetic variants associated with TCC using protein quantitative trait loci analysis of exome sequencing data. Methods:We sampled 415 VTE cases and 848 age-and sex-matched controls from a population-based cohort, the Tromsø study. Logistic regression models were used to calculate odds ratios with 95% confidence intervals for VTE across quartiles of plasma levels of TCC. Whole exome sequencing was conducted using the Agilent SureSelect 50 Mb capture kit.Results: The risk of VTE increased across increasing quartiles of plasma TCC, particularly for unprovoked VTE. Participants with TCC in the highest quartile (>1.40 complement arbitrary units/mL) had an odds ratio for unprovoked VTE of 1.74 (95% confidence interval: 1.10-2.78) compared with those with TCC in the lowest quartile (≤0.80 complement arbitrary units/mL) in analyses adjusted for age, sex, and body mass index. A substantially higher risk for VTE was observed in samples taken shortly before VTE event. We found no association between genome-wide or complementrelated gene variants and plasma levels of TCC. Conclusions:We found that high levels of plasma TCC were associated with VTE risk, and unprovoked events in particular. There was no genome-wide association between gene variants and plasma levels of TCC. K E Y W O R D S complement system, protein quantitative trait loci analysis, terminal complement complex, venous thromboembolism, whole exome sequencing | 935 HØILAND et AL. S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Høiland II, Liang RA, Braekkan SK, Pettersen K, Ludviksen JK, Latysheva N, et al. Complement activation assessed by the plasma terminal complement complex and future risk of venous thromboembolism. J Thromb Haemost. 2019;17:934-943. https ://doi.

Background Most tissue factor (TF) activity assays are based on measurement of factor X (FX) activation by TF in the presence of factor VII (FVII)/FVIIa. This requires long incubation, which may result in TF‐independent activity of FX and inaccurate measurement of TF activity. Aim To develop a sensitive and specific TF activity assay, which does not register a non‐specific TF activity, using commercial coagulation factors. Methods Tissue factor activity was measured based on the ability of TF to accelerate the activation of FX by FVIIa in the presence of factor V (FV)/Va, prothrombin, and phospholipids. Following 4 min incubation at 37°C, TF activity was quantified in test samples of different nature by thrombin generation using a chromogenic substrate. Results The TF activity assay proved high sensitivity (low fM range) and specificity, assessed by neutralization of TF activity by anti‐TF antibody and the use of FVIIai. TF activity was detected in extracellular vesicles (EVs) derived from HAP1‐TF+cells, while no activity was measured in EVs from HAP1‐TF/KO cells. The assay was applicable for measurement of TF activity on the surface of live endothelial cells and monocytes activated in vitro, and cell lysates. Infusion of low dose lipopolysaccharide (2 ng/kg bodyweight endotoxin) caused a transient 8‐fold increase (peaked at 4 h) in TF activity in EVs isolated from plasma of healthy volunteers. Conclusion Our assay provides a fast, sensitive, and specific measurement of TF activity. It reliably quantifies TF activity on cell surface, cell lysate, and isolated EVs. The assay can be used for laboratory and clinical research.

Growing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL). Current commercial PPL-dependent clotting assays use chemically phospholipid depleted plasma to measure PPL activity. The purpose of our study was to modify the PPL assay by substituting the chemically phospholipid depleted plasma with PPL depleted plasma obtained by ultracentrifugation This in order to get readily access to a sensitive and reliable assay to measure PPL activity in human plasma and cell supernatants. The performance of the assay was tested, including the influence of individual coagulation factors and postprandial lipoproteins and compared to a commercial PPL assay (STA-Procoag-PPL). The two PPL assays displayed similar sensitivity to exogenously added standardized phospholipids. The PPL activity measured by the modified assay strongly correlates with the results from the commercial assay. The intraday- and between-days coefficients of variation ranged from 2–4% depending on the PPL activity in the sample. The modified PPL assay was insensitive to postprandial lipoprotein levels in plasma, as well as to tissue factor (TF) positive EVs from stimulated whole blood. Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF+ EVs.

The article elucidates the physical mechanism behind the generation of superior-contrast and high-resolution label-free images using an optical waveguide. Imaging is realized by employing a high index contrast multi-moded waveguide as a partially coherent light source. The modes provide near-field illumination of unlabeled samples, thereby repositioning the higher spatial frequencies of the sample into the far-field. These modes coherently scatter off the sample with different phases and are engineered to have random spatial distributions within the integration time of the camera. This mitigates the coherent speckle noise and enhances the contrast (2–10) × as opposed to other imaging techniques. Besides, the coherent scattering of the different modes gives rise to fluctuations in intensity. The technique demonstrated here is named chip-based Evanescent Light Scattering (cELS). The concepts introduced through this work are described mathematically and the high-contrast image generation process using a multi-moded waveguide as the light source is explained. The article then explores the feasibility of utilizing fluctuations in the captured images along with fluorescence-based techniques, like intensity-fluctuation algorithms, to mitigate poor-contrast and diffraction-limited resolution in the coherent imaging regime. Furthermore, a straight waveguide is demonstrated to have limited angular diversity between its multiple modes and therefore, for isotropic sample illumination, a multiple-arms waveguide geometry is used. The concepts introduced are validated experimentally via high-contrast label-free imaging of weakly scattering nanosized specimens such as extra-cellular vesicles (EVs), liposomes, nanobeads and biological cells such as fixed and live HeLa cells.