Journal: EBioMedicine

Article Title: Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation

doi: 10.1016/j.ebiom.2021.103382

Figure Lengend Snippet: FXII is activated in COVID-19 lungs. (a) Representative immunofluorescence of vibratome sections from FXII-increased COVID-19 and Acute Respiratory Distress Syndrome (ARDS) control lungs using antibodies against FXIIa (3F7, green or grey in single channel images) and Collagen I (magenta). White arrows indicate FXIIa signal within the microvasculature. Alveolar space (AS), parenchyma (PA). (b) Representative immunoblot of COVID-19 and healthy donor plasma samples blotted for FXII (GAHu/FXII, Nordic MUbio, Netherlands). The antibody detects FXII zymogen and cleaved, activated FXII (FXIIa). As negative control FXII deficient plasma was used. (c) Quantification of FXIIa in COVID-19 plasma (n=19) and age- and sex-matched healthy donor plasma (n=15). FXIIa was normalized to plasma albumin levels. Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test. Scale bar: 100 µm top/10 µm middle/50 µm bottom panel (a). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following antibodies were used: anti-neutrophil elastase (5µg/ml, ab21595, Abcam), anti-chromatin (anti-histone H2A/H2B/DNA-complex, 5µg/ml, Davids Biotechnologie GmbH ), anti-Collagen I (5µg/ml, Ab34710, Abcam), anti-fibrin (5µg/ml, MABS2155, Merck), AlexaFluor488-conjugated anti-FXIIa (10µg/ml, 3F7, CSL Limited), anti-citrullinated histone 3 (1:500, ab5103, Abcam).

Techniques: Immunofluorescence, Western Blot, Negative Control, Two Tailed Test

Journal: EBioMedicine

Article Title: Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation

doi: 10.1016/j.ebiom.2021.103382

Figure Lengend Snippet: NETs and FXIIa colocalize in COVID-19 lungs. (a) High magnification immunofluorescence images of NETs in the lung parenchyma and the alveolar spaces using antibodies against NE (magenta) and chromatin (green or grey in single channel images). (b) Representative immunofluorescence of vibratome sections of COVID-19 and control (CTRL) lungs using antibodies against activated FXII (3F7, green or grey in single channel images) and histone 3 (H3cit) (magenta or grey in single channel images). (c) FXIIa pixel intensity was measured in n=107 NET-positive areas from three COVID-19 lungs and n=35 NET-positive areas from three CTRL lungs. Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test. Scale bar: 10 µm (a,b). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following antibodies were used: anti-neutrophil elastase (5µg/ml, ab21595, Abcam), anti-chromatin (anti-histone H2A/H2B/DNA-complex, 5µg/ml, Davids Biotechnologie GmbH ), anti-Collagen I (5µg/ml, Ab34710, Abcam), anti-fibrin (5µg/ml, MABS2155, Merck), AlexaFluor488-conjugated anti-FXIIa (10µg/ml, 3F7, CSL Limited), anti-citrullinated histone 3 (1:500, ab5103, Abcam).

Techniques: Immunofluorescence, Two Tailed Test

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Cationic Zinc is Required for Factor XII Recruitment and Activation by Stimulated Platelets and for Thrombus Formation in vivo

doi: 10.1111/jth.14964

Figure Lengend Snippet: (A) Thrombin generation was measured after platelet rich plasma (PRP) was stimulated with 50 μM of the PAR-1 receptor agonist peptide SFLLRN in the presence of either non-immune control antibody (SFLLRN) or inhibitory antibodies against FXIIa (3F7, left, N=12 replicates), factor VIIa (middle, N=12 replicates), or tissue factor (right, N=15 replicates). Each experiment was performed independently two times with 9 replicates per condition. (B) PRP was stimulated with 50 μM SFLLRN in the presence of increasing concentrations of the Zn2+-specific chelator CaEDTA and FXIIa generation was recorded (left, N=9 replicates per condition). P-selectin (CD62P) expression was measured in resting and SFLLRN-stimulated platelets in the presence or absence of 40 mM CaEDTA (right, N=3 replicates per condition). (C) By comparison, the activated partial thromboplastin time (aPTT) was recorded in the presence of increasing concentrations of CaEDTA, N=3 replicates per condition. **P<0.05 compared to activated platelet control by Kruskal-Wallis test with Dunn’s post-test for multiple comparisons.

Article Snippet: Anti-FXIIa antibody 3F7 was custom manufactured by Viva Biotech (Shanghai, China) based on complementarity-determining region (CDR) sequences obtained from the published international patent (WO2014207199A1), and its performance was confirmed by aPTT assay ( Figure S1 ).

Techniques: Clinical Proteomics, Control, Expressing, Comparison

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Cationic Zinc is Required for Factor XII Recruitment and Activation by Stimulated Platelets and for Thrombus Formation in vivo

doi: 10.1111/jth.14964

Figure Lengend Snippet: Human umbilical vein endothelial cells were stimulated with 10 ng/ml TNF-α, and thrombin generation was recorded in the presence of inhibitory antibodies (10 μg/ml) or calcium EDTA (40 mM) as indicated. Antibody 3F7 was used for FXIIa inhibition. N=4 replicates per condition, **P<0.05 for comparison to TNF-α condition.

Article Snippet: Anti-FXIIa antibody 3F7 was custom manufactured by Viva Biotech (Shanghai, China) based on complementarity-determining region (CDR) sequences obtained from the published international patent (WO2014207199A1), and its performance was confirmed by aPTT assay ( Figure S1 ).

Techniques: Inhibition, Comparison

Journal: EBioMedicine

Article Title: Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation

doi: 10.1016/j.ebiom.2021.103382

Figure Lengend Snippet: FXII is activated in COVID-19 lungs. (a) Representative immunofluorescence of vibratome sections from FXII-increased COVID-19 and Acute Respiratory Distress Syndrome (ARDS) control lungs using antibodies against FXIIa (3F7, green or grey in single channel images) and Collagen I (magenta). White arrows indicate FXIIa signal within the microvasculature. Alveolar space (AS), parenchyma (PA). (b) Representative immunoblot of COVID-19 and healthy donor plasma samples blotted for FXII (GAHu/FXII, Nordic MUbio, Netherlands). The antibody detects FXII zymogen and cleaved, activated FXII (FXIIa). As negative control FXII deficient plasma was used. (c) Quantification of FXIIa in COVID-19 plasma (n=19) and age- and sex-matched healthy donor plasma (n=15). FXIIa was normalized to plasma albumin levels. Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test. Scale bar: 100 µm top/10 µm middle/50 µm bottom panel (a). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Together with CSL Behring, we have previously demonstrated that targeting FXIIa with the neutralizing antibody 3F7 provides thrombo-protection and impedes bradykinin production in a preclinical setting .

Techniques: Immunofluorescence, Control, Western Blot, Clinical Proteomics, Negative Control, Two Tailed Test

Journal: EBioMedicine

Article Title: Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation

doi: 10.1016/j.ebiom.2021.103382

Figure Lengend Snippet: NETs and FXIIa colocalize in COVID-19 lungs. (a) High magnification immunofluorescence images of NETs in the lung parenchyma and the alveolar spaces using antibodies against NE (magenta) and chromatin (green or grey in single channel images). (b) Representative immunofluorescence of vibratome sections of COVID-19 and control (CTRL) lungs using antibodies against activated FXII (3F7, green or grey in single channel images) and histone 3 (H3cit) (magenta or grey in single channel images). (c) FXIIa pixel intensity was measured in n=107 NET-positive areas from three COVID-19 lungs and n=35 NET-positive areas from three CTRL lungs. Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test. Scale bar: 10 µm (a,b). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Together with CSL Behring, we have previously demonstrated that targeting FXIIa with the neutralizing antibody 3F7 provides thrombo-protection and impedes bradykinin production in a preclinical setting .

Techniques: Immunofluorescence, Control, Two Tailed Test