Journal: medRxiv

Article Title: Elevated Angiopoietin-2 inhibits thrombomodulin-mediated anticoagulation in critically ill COVID-19 patients

doi: 10.1101/2021.01.13.21249429

Figure Lengend Snippet: (A) Plasma ANGPT2 concentrations for healthy controls (HC), recovered (Rec) and deceased (Dec) patients at day 1-4 and day 10-14 after admission. (B, C) Kaplan Meier plots with Log-rank test show that ANGPT2 levels are higher in non-recovering patients both at admission and when including all time-points and can predict mortality. (D) Plasma ANGPT1 was minimally affected. (E) Clinical data and measured parameters were analyzed by Pearson correlation test (Spearman for SAPS-2 and SOFA score) and presented in a heatmap for correlation (blue) or inverse correlation (red). A summary of ANGPT2 comparisons is presented in , and all data in . (F) ANGPT2 is higher in patients with acute kidney injury (AKI) compared to non-AKI patients. (G) ANGPT2 is higher in patients with MA >69. (H) Thromboelastography (TEG) measurements for MA, other TEG measurements can be seen in . (I, J) Immunoprecipitation and quantification of thrombomodulin (TM) from plasma in controls and COVID-19 patients show binding of ANGPT2. Full uncut blots available in . (K, L) Plasma concentrations for VWF and ADAMTS13, respectively. Data shown as shown as geometric mean ± 95% CI (A, D, F-H, J-L). # # # # p<0.0001 vs. HC, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. p-value from one-way ANOVA with Bonferroni post hoc test (A, D, H, K, L), and t-test (F, G, J) on log transformed data. TEG thromboelastography; MA maximal amplitude

Article Snippet: Pooled plasma from healthy controls was incubated with the same volume of 6 mM CaCl 2 with 0.2 U/ml thrombin (T8885, Sigma), 1000 ng/ml of recombinant thrombomodulin (3947-PA-010, R&D Systems), ANGPT2 (ab220589, Abcam), ANGPT1 (ab69492, Abcam), IgG (ab219660, Abcam) at 37°C for 30 minutes.

Techniques: Clinical Proteomics, Immunoprecipitation, Binding Assay, Transformation Assay

Journal: medRxiv

Article Title: Elevated Angiopoietin-2 inhibits thrombomodulin-mediated anticoagulation in critically ill COVID-19 patients

doi: 10.1101/2021.01.13.21249429

Figure Lengend Snippet: (A) Tail bleeding time 15 min after i.p. injection of His tagged ANGPT2 (A2), and ANGPT1 (A1) at indicated doses. Albumin or IgG was injected in controls. (B) Plasma concentrations of ANGPT2 after i.p injection. (C, D) Immunoprecipitation and quantification of thrombomodulin (TM) from lung lysates in injected mice with blotting for His tag and TM. (G, H) Tail bleeding time in Angpt2 iECKO mice and Tie2 iECKO mice, respectively. Data presented as geometric mean ± 95% CI. * p<0.05, ** p<0.01, **** p<0.0001. p-value from one-way ANOVA with Bonferroni post hoc test (A, B, D), and t-test (G, H) on log transformed data.

Article Snippet: Pooled plasma from healthy controls was incubated with the same volume of 6 mM CaCl 2 with 0.2 U/ml thrombin (T8885, Sigma), 1000 ng/ml of recombinant thrombomodulin (3947-PA-010, R&D Systems), ANGPT2 (ab220589, Abcam), ANGPT1 (ab69492, Abcam), IgG (ab219660, Abcam) at 37°C for 30 minutes.

Techniques: Injection, Clinical Proteomics, Immunoprecipitation, Transformation Assay

Journal: medRxiv

Article Title: Elevated Angiopoietin-2 inhibits thrombomodulin-mediated anticoagulation in critically ill COVID-19 patients

doi: 10.1101/2021.01.13.21249429

Figure Lengend Snippet: TEG curve from one of the donors showing thrombomodulin (TM) dependent increase in reaction time (R) and decreased maximal amplitude (MA), which is inhibited by ANGPT2 (A). TEG analysis of individual donor blood with addition of 1000 ng/ml thrombomodulin (TM) and 1000 ng/ml ANGPT2. Foldchange graphs from TEG analysis for R (B) and MA (C). Group results from these experiments can be found in . Thrombomodulin dependent formation of APC can be inhibited with ANGPT2 or ANGPT1 (D). Data presented as geometric mean ± 95% CI. ** p<0.01, *** p<0.001, **** p<0.0001. p-value from one-way ANOVA with Bonferroni post hoc test on log transformed data. TEG thromboelastography

Article Snippet: Pooled plasma from healthy controls was incubated with the same volume of 6 mM CaCl 2 with 0.2 U/ml thrombin (T8885, Sigma), 1000 ng/ml of recombinant thrombomodulin (3947-PA-010, R&D Systems), ANGPT2 (ab220589, Abcam), ANGPT1 (ab69492, Abcam), IgG (ab219660, Abcam) at 37°C for 30 minutes.

Techniques: Transformation Assay

Journal: medRxiv

Article Title: Elevated Angiopoietin-2 inhibits thrombomodulin-mediated anticoagulation in critically ill COVID-19 patients

doi: 10.1101/2021.01.13.21249429

Figure Lengend Snippet: A schematic overview of Angiopoietin signaling in normal coagulation and in hypercoagulation with high ANGPT2. In addition to ANGPT2 inhibition of thrombomodulin mediated anticoagulation several other endothelial functions are disturbed by high ANGPT2.

Article Snippet: Pooled plasma from healthy controls was incubated with the same volume of 6 mM CaCl 2 with 0.2 U/ml thrombin (T8885, Sigma), 1000 ng/ml of recombinant thrombomodulin (3947-PA-010, R&D Systems), ANGPT2 (ab220589, Abcam), ANGPT1 (ab69492, Abcam), IgG (ab219660, Abcam) at 37°C for 30 minutes.

Techniques: Coagulation, Inhibition

Journal: Journal of Thrombosis and Haemostasis

Article Title: Defective fibrin deposition and thrombus stability in Bambi −/− mice are mediated by elevated anticoagulant function

doi: 10.1111/jth.14593

Figure Lengend Snippet: A, B, Increased thrombomodulin and TFPI expression levels in Bambi −/− mice. Representative Western blot of A, TFPI and B, thrombomodulin expression in Bambi +/+ and Bambi −/− lung homogenates. Protein levels were quantified using Image Lab 5.2.1 software (Biorad), normalized against GAPDH controls and expressed as relative intensities ( TFPI or TM / GAPDH ). Values are given as mean ± SEM ( n ≥ 7 mice per genotype from at least two Western blots). Statistical analysis was performed using unpaired Student t test; * P < .05; ** P < .01. Molecular weights from protein standards are indicated in kilodaltons on each Western blot. C, Bambi +/+ and Bambi −/− MLEC were incubated with 100 nM human protein C in the presence of Ca 2+ and activated by 2 nM thrombin. The reactions were stopped at the indicated times by addition of an excess of hirudin (100 nM). The APC generation was quantified by determining the rate of chromogenic substrate S‐2366 (0.5 mM) cleavage at 405 nm and using an APC standard curve generated in parallel for each experiment (cf. Figure ). The APC generation was normaliszed to the number of cells and maximal APC activity for each assay. When indicated (+α‐ TM ), Bambi +/+ and Bambi −/− MLEC were incubated for 30 min with a goat antimouse thrombomodulin antibody (50 nM) before addition of protein C and thrombin to the wells and APC activity was determined after 60 min. Values are given as mean ± SEM ( n = two separate isolations). Statistical analysis was performed using two‐way ANOVA followed by Bonferroni posttests; * P < .05 ** P < .01. APC, activated protein C; GADPH, Glyceraldehyde 3‐phosphate dehydrogenase; MLEC, mouse lung endothelial cell; TFPI, tissue factor pathway inhibitor

Article Snippet: Thrombin generation was assessed by calibrated automated thrombography (CAT) using a Fluoroskan Ascent FL plate reader (Thermo, Paisley, UK) in combination with Thrombinoscope software (Synapse BV) as described previously., Briefly, the generation of thrombin was quantified in human plasma (80 μL per well) supplemented with 4 μM phospholipid vesicles in the presence and absence of 10 nM recombinant mouse thrombomodulin (R&D, Bio‐Techne) and 10 to 100 nM goat antimouse thrombomodulin antibody (R&D, Bio‐Techne).

Techniques: Expressing, Western Blot, Software, Incubation, Generated, Activity Assay

Journal: Journal of Thrombosis and Haemostasis

Article Title: Defective fibrin deposition and thrombus stability in Bambi −/− mice are mediated by elevated anticoagulant function

doi: 10.1111/jth.14593

Figure Lengend Snippet: Inhibiting thrombomodulin and TFPI in vivo in Bambi −/− mice restores thrombus stability and fibrin generation in the laser‐induced thrombosis model. A, Representative composite fluorescence images of platelets (green) and fibrin (red) with bright field images after the laser injury in cremaster arterioles of Bambi +/+ and Bambi −/− mice injected with control goat immunoglobulin G (IgG), goat anti mouse thrombomodulin antibody (α‐ TM ), goat antimouse TFPI (α‐ TFPI ), or a combination of both α‐ TM and α‐ TFPI . Platelets and fibrin were visualized by injecting rat anti‐ GPI bβ‐DyLight 488 antibody and fibrinogen‐A647, respectively. Scale bar represents 10 μm. B, Graph represents median integrated fluorescence intensity ( IFI ) from platelets (AU, arbitrary unit) as a function of time after the injury. Thrombi from Bambi +/+ and Bambi −/− mice injected with control goat IgG are overlaid with thrombi from mice injected with α‐ TM (Bi), α‐ TFPI (Bii), or a combination of both α‐ TM and α‐ TFPI antibodies (Biii) (43 thrombi in seven Bambi +/+ mice (+ Goat IgG); 45 thrombi in six Bambi −/− mice (+ Goat IgG); 25 thrombi in three Bambi +/+ mice (+ α‐ TM ); 27 thrombi in three Bambi −/− mice (+ α‐ TM ); 21 thrombi in two Bambi +/+ mice (+ α‐ TFPI ); 28 thrombi in three Bambi −/− mice (+ α‐ TFPI ); 20 thrombi in two Bambi +/+ mice (+ α‐ TFPI + α‐ TM ); 20 thrombi in two Bambi −/− mice (+ α‐ TFPI + α‐ TM ). The area under the curve of fluorescence intensity over 180 s was analyzed using an unpaired Mann Whitney test; ns: P > .05. C, Number of emboli (>25% maximal thrombus size) counted during 3 min after laser injury. Results are shown as mean ± SEM . Statistical analysis was performed using one‐way ANOVA with Dunnett's multiple comparisons test; versus Bambi +/+ in Ci. or versus Bambi −/− in Cii. * P < .05. D, Graph represents fibrin(ogen)‐ IFI ( AU ) as a function of time after the laser injury. Di. Bambi +/+ mice injected with Goat IgGs, α‐ TM , α‐ TFPI , α‐ TFPI and α‐ TM antibodies. Dii. Bambi −/− mice injected with Goat IgGs, α‐ TM , α‐ TFPI , α‐ TFPI and α‐ TM antibodies. The area under the curve of fluorescence intensity over 180 s was analyzed using an unpaired Mann Whitney test; ** P < .01; * P < .05; ns: P > .05. See also Movies and for better visualization of the differences in thrombus stability between Bambi +/+ and Bambi −/− mice. TFPI, tissue factor pathway inhibitor

Article Snippet: Thrombin generation was assessed by calibrated automated thrombography (CAT) using a Fluoroskan Ascent FL plate reader (Thermo, Paisley, UK) in combination with Thrombinoscope software (Synapse BV) as described previously., Briefly, the generation of thrombin was quantified in human plasma (80 μL per well) supplemented with 4 μM phospholipid vesicles in the presence and absence of 10 nM recombinant mouse thrombomodulin (R&D, Bio‐Techne) and 10 to 100 nM goat antimouse thrombomodulin antibody (R&D, Bio‐Techne).

Techniques: In Vivo, Fluorescence, Injection, MANN-WHITNEY