Journal: bioRxiv

Article Title: Neutrophil extracellular traps block endogenous and intravenous thrombolysis-induced fibrinolysis in large vessel occlusion acute ischemic stroke

doi: 10.1101/2025.03.28.646062

Figure Lengend Snippet: Intravenous thrombolysis increases tissue-type plasminogen activator content in acute ischemic stroke thrombi despite therapeutic failure. A-C. Comparison of tissue-type plasminogen activator (tPA) (A), D-dimer (B), and plasminogen (C) content in thrombi from patients with acute ischemic stroke due to large vessel occlusion having received intravenous thrombolysis (IVT) or not (no-IVT) before endovascular thrombectomy. D. Western blot analysis of tPA in homogenates of 5 no-IVT (Thr1 to 5) and 5 IVT (Thr6 to 10) thrombi in non-reducing (upper panel) and reducing (lower panel) conditions. Note that in both no-IVT and IVT thrombi, tPA is mainly found under the form of 3 high-molecular-weight complexes (indicated by arrows and labeled a to c in the upper panel), from which it could be dissociated as free single chain tPA under reducing conditions (arrow and labeled d in the lower panel).

Article Snippet: The membranes were probed with antibodies to α2AP (ab150414, abcam), plasminogen (PA5-14196, Invitrogen), tPA (NBP2-20648, Novus Biologicals), and histone H1 (SC-67324, Santa Cruz), revealed with Dylight 800- and Dylight 680-conjugated secondary antibodies using a LI-COR Odyssey® imaging system.

Techniques: Comparison, Western Blot, High Molecular Weight, Labeling

Journal: bioRxiv

Article Title: Neutrophil extracellular traps block endogenous and intravenous thrombolysis-induced fibrinolysis in large vessel occlusion acute ischemic stroke

doi: 10.1101/2025.03.28.646062

Figure Lengend Snippet: A. Representative images of immunofluorescent localization of tissue-type plasminogen activator (tPA, green) in thrombi from IS patients. Top panels: accumulation of tPA in association with compact fibrin (red) in the thrombus outer shell (arrows). Middle panels: tPA bound to fibrin fibers in a red blood cell-rich area of the thrombus core and with compact fibrin (arrows). Lower panels: the white arrows indicate tPA associated with clusters of dense fibrin entangled with extracellular DNA (cyan) close to the thrombus surface. The images shown were acquired in a thrombus from a patient having received intravenous thrombolysis prior to EVT and are representative of 10 different thrombi.

Article Snippet: The membranes were probed with antibodies to α2AP (ab150414, abcam), plasminogen (PA5-14196, Invitrogen), tPA (NBP2-20648, Novus Biologicals), and histone H1 (SC-67324, Santa Cruz), revealed with Dylight 800- and Dylight 680-conjugated secondary antibodies using a LI-COR Odyssey® imaging system.

Techniques:

Journal: bioRxiv

Article Title: Neutrophil extracellular traps block endogenous and intravenous thrombolysis-induced fibrinolysis in large vessel occlusion acute ischemic stroke

doi: 10.1101/2025.03.28.646062

Figure Lengend Snippet: A. Schematic representation of the ex vivo thrombolysis assay performed with thrombi from stroke patients that had or had not received intravenous thrombolysis with tissue-type plasminogen activator (tPA) prior to mechanical thrombectomy. B. Mean thrombus weight evolution over 1 hour of ex vivo thrombolysis initiated by the addition of plasminogen (100 µg/mL), in the presence or absence of DNase 1 (pulmozyme, 100 µg/mL), between thrombi from patients having received (IVT) or not (no-IVT) intravenous thrombolysis prior to thrombectomy. Error bars indicate standard error of the mean. C. Comparison of the reduction in thrombus weight after 60 min of ex vivo incubation of acute ischemic thrombi (AIS) with plasminogen alone or combined with DNase 1 between thrombi recovered from patients having received tPA (IVT) or not (no-IVT) prior to thrombectomy. Results are expressed as a percentage relative to the initial thrombus weight. D. Comparison of the reduction in thrombus weight after 60 min of ex vivo incubation of AIS thrombi with plasminogen and DNase 1 according to the type of recombinant tPA used for IVT. Results are expressed as a percentage relative to the initial thrombus weight. Each dot represents a different thrombus.

Article Snippet: The membranes were probed with antibodies to α2AP (ab150414, abcam), plasminogen (PA5-14196, Invitrogen), tPA (NBP2-20648, Novus Biologicals), and histone H1 (SC-67324, Santa Cruz), revealed with Dylight 800- and Dylight 680-conjugated secondary antibodies using a LI-COR Odyssey® imaging system.

Techniques: Ex Vivo, Comparison, Incubation, Recombinant

Journal: bioRxiv

Article Title: Neutrophil extracellular traps block endogenous and intravenous thrombolysis-induced fibrinolysis in large vessel occlusion acute ischemic stroke

doi: 10.1101/2025.03.28.646062

Figure Lengend Snippet: A. Schematic representation of sequential ex vivo thrombolysis in which acute ischemic stroke (AIS) thrombi were cut in half and first incubated with DNase 1 alone (pulmozyme, 100 µg/mL), before being subsequently treated with tissue-type plasminogen activator (tPA, alteplase, 1 µg/mL) and plasminogen (100 µg/mL or 1 µM). B. Comparison of mean thrombus weight evolution in response to tPA and plasminogen added following a pretreatment with either DNase 1 or vehicle. n = 16 thrombi, *** p < 0.0005 for the comparison between the two groups at each time point. C. Paired comparison of thrombus weight after 60 min of ex vivo thrombolysis triggered by addition of tPA and plasminogen after a pretreatment of AIS thrombi with either DNase 1 or corresponding vehicle. Each dot corresponds to a different thrombus fragment, n = 16 thrombi, each cut in half for paired comparison of both conditions.

Article Snippet: The membranes were probed with antibodies to α2AP (ab150414, abcam), plasminogen (PA5-14196, Invitrogen), tPA (NBP2-20648, Novus Biologicals), and histone H1 (SC-67324, Santa Cruz), revealed with Dylight 800- and Dylight 680-conjugated secondary antibodies using a LI-COR Odyssey® imaging system.

Techniques: Ex Vivo, Incubation, Comparison

Journal: CNS Neuroscience & Therapeutics

Article Title: Asparagine Endopeptidase Inhibition Attenuates Tissue Plasminogen Activator‐Induced Brain Hemorrhagic Transformation After Ischemic Stroke

doi: 10.1111/cns.70345

Figure Lengend Snippet: AEP KO ameliorates tPA‐associated hemorrhagic transformation in the stroke mouse model. (A, B) Representative images of hematoma in brain slices and quantification of hemoglobin levels. Data are presented as mean ± SEM and statistical analyses are performed using Welch test followed by Dunnett T3 multiple comparisons test were applied since the P value of Levene test < 0.05. (C) Recorded brain water content to assess brain edema at 24 h after delayed tPA administration. Data are presented as mean ± SEM and statistical analysis is performed using one‐way ANOVA test followed by Tukey's multiple comparisons test. (D) ELISA for tPA activity detection. Data are presented as mean ± SEM and statistical analyses are performed using Welch test followed by Dunnett T3 multiple comparisons test were applied since the P value of Levene test < 0.05. (E) Representative images of HE staining in the cortical hematoma area (scale bar = 50 μm). (A), (C) n = 6, (D) n = 4 per group. Normality and variance are assessed via Shapiro‐Wilk test and Levene's test, respectively. * P < 0.05, ** P < 0.01.

Article Snippet: A human Tissue‐type Plasminogen Activator ELISA kit (E‐EL‐H2106, Elabscience) was used for detection.

Techniques: Transformation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Asparagine Endopeptidase Inhibition Attenuates Tissue Plasminogen Activator‐Induced Brain Hemorrhagic Transformation After Ischemic Stroke

doi: 10.1111/cns.70345

Figure Lengend Snippet: The 7,8‐DHF prodrug R13 confers protection in mice against neurological dysfunction, hemorrhagic transformation, and BBB disruption after cerebral ischemia and tPA treatment. Mice were given R13 (dissolved in 5% DMSO/0.5% methylcellulose) at a dose of 21.8 mg/kg/day, 7 days per week, for 2 weeks by gavage, followed by MCAO/R. Surgery and delayed tPA treatment. (A, B) Representative images of TTC staining and the corresponding proportion of the infarct area. (C, D) Representative images of hematoma in brain slices and quantification of hemoglobin levels. (E, F) Representative images and leakage quantification of Evans Blue staining. (G, H) Longa Scores and Corner Turn tests used for sensorimotor function examination. (I) ELISA for tPA activity detection. (A), (C), (F), (I) n = 4, (G, H) n = 20 per group. Data are presented as mean ± SEM, and statistical analyses are performed using one‐way ANOVA test followed by Tukey's multiple comparisons test when P value of Levene test > 0.05 or Welch test followed by Dunnett T3 multiple comparisons test when the P value of Levene test < 0.05. Normality and variance are assessed via Shapiro‐Wilk test and Levene's test, respectively. * P < 0.05, ** P < 0.01.

Article Snippet: A human Tissue‐type Plasminogen Activator ELISA kit (E‐EL‐H2106, Elabscience) was used for detection.

Techniques: Transformation Assay, Disruption, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay