Journal: Scientific reports

Article Title: GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura.

doi: 10.1038/s41598-024-80674-x

Figure Lengend Snippet: Fig. 1. The process and outcomes of candidate screening and the pharmacokinetic profile of candidates. (a) The diagram shows the binding domains of anti-ADAMTS13 neutralizing antibodies (Nabs). (b) The strategy and process of candidate screening. (c) The specific activity of selected candidates in the media following transient expression (n = 1 or 2). (d) The relative residual activity of selected candidates in the presence of 9 Nabs (n = 1 or 2). (e) The plasma concentration profiles after the intravenous administration of the selected candidates or controls (MDTCS or MDTCS-Fc) at the dose level of 160 IU/kg. Each point represents the mean and standard error of the mean (n = 4 mice per time point).

Article Snippet: The recombinant human ADAMTS13 (full-length) protein (rhADAMTS13 FL; R&D Systems, Minnesota, USA) served as the antigen.

Techniques: Binding Assay, Activity Assay, Expressing, Clinical Proteomics, Concentration Assay

Journal: Scientific reports

Article Title: GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura.

doi: 10.1038/s41598-024-80674-x

Figure Lengend Snippet: Fig. 4. Autoantibody escaping ability of GC1126A using the iTTP patient’s plasma. (a) Relative residual activity of ADAMTS13 and GC1126A in the presence of autoantibodies from iTTP patient’s plasma (n = 2). Each patient’s plasma was diluted to inhibitory antibody concentrations of 0.5, 1, 2, and 3 BU/ml. Each diluted plasma was mixed with the same molar concentration (3 nM) of ADAMTS13 or GC1126A. Relative activity is the ratio of activity remaining after neutralizing antibodies in the patient’s plasma to activity when each substance is free of neutralizing antibodies. (b) Binding level of autoantibodies from patients with iTTP to ADAMTS13 and GC1126A. Statistical significance was determined using the one-way analysis of variance followed by Tukey’s test (α = 0.05). Each bar represents the mean and standard error of the mean (n = 3).

Article Snippet: The recombinant human ADAMTS13 (full-length) protein (rhADAMTS13 FL; R&D Systems, Minnesota, USA) served as the antigen.

Techniques: Clinical Proteomics, Activity Assay, Concentration Assay, Binding Assay

Journal: Scientific reports

Article Title: GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura.

doi: 10.1038/s41598-024-80674-x

Figure Lengend Snippet: Fig. 5. Relationship between the anti-ADAMTS13 inhibitor titer and GC1126A EC50. The distribution of the amount of GC1126A required to achieve 0.5 IU/ml activity (EC50) for different patient samples at inhibitor titers of 0.6–0.9 BU/ml (n = 4), 1 BU/ml (n = 23), 3 BU/ml (n = 15), 6 BU/ml (n = 9), and 9 BU/ml (n = 5) (the observed data are shown with closed circles). The estimated regression line and the 95% confidence interval are depicted in solid and dotted lines, respectively. The linear regression analysis yielded the following relationship: EC50 (µg/mL) = 0.0347 + 0.0396 × anti-ADAMTS13 inhibitor titer (BU/mL) (R2 = 0.5842).

Article Snippet: The recombinant human ADAMTS13 (full-length) protein (rhADAMTS13 FL; R&D Systems, Minnesota, USA) served as the antigen.

Techniques: Activity Assay

Journal: Scientific reports

Article Title: GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura.

doi: 10.1038/s41598-024-80674-x

Figure Lengend Snippet: Fig. 6. Comparison of therapeutic efficacy of GC1126A, rh WT-ADAMTS13, and caplacizumab in mitigating platelet count reduction in the iTTP-mimic mouse model. (a) Study design for therapeutic efficacy comparison. (b) Platelet count results. (c) Residual activity of ADAMTS13 in the plasma of an iTTP-mimic mouse model. Values below the limit (0.03 IU/mL) are also not present on the graph. (d) Inhibitory antibody levels (BU levels). Values below the limit (0.42 BU/mL) are also not present on the graph. (b)–(d) Outliers were identified using the Grubbs’ test (α = 0.05) and excluded from each group. Each bar represents the mean and standard error of the mean (n = 4 mice per group).

Article Snippet: The recombinant human ADAMTS13 (full-length) protein (rhADAMTS13 FL; R&D Systems, Minnesota, USA) served as the antigen.

Techniques: Comparison, Drug discovery, Activity Assay, Clinical Proteomics

Journal: Scientific reports

Article Title: GC1126A, a novel ADAMTS13 mutein, evades autoantibodies in immune-mediated thrombotic thrombocytopenic purpura.

doi: 10.1038/s41598-024-80674-x

Figure Lengend Snippet: Fig. 7. Comparison of therapeutic efficacy of GC1126A, rh WT-ADAMTS13, and caplacizumab in restoring severely reduced platelet counts in the iTTP-mimic mouse model. (a) Study design for therapeutic efficacy comparison. (b) Platelet count results. Outliers were identified using the Grubbs’ test (α = 0.05) and excluded from each group. Each bar represents the mean and standard error of the mean (n = 3–10 mice per group).

Article Snippet: The recombinant human ADAMTS13 (full-length) protein (rhADAMTS13 FL; R&D Systems, Minnesota, USA) served as the antigen.

Techniques: Comparison, Drug discovery

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 1. Immunohistochemical analysis of CX3CL1 expression in human colorectal cancer. (A) Representative immunoreactivity of CX3CL1 in normal colonic mucosa, precancerous lesion (adenoma), and colorectal cancer. Original magnification 320. (B) Expression scores of CX3CL1 in all tested samples (normal colonic mucosa, n = 18; adenoma, n = 18; colorectal cancer, n = 100). CX3CL1 increases in adenoma and carcinoma, but decreases in stage III tumors. Red bars indicate median value. Kruskal– Wallis and Dunn’s posttest, *p , 0.05.

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: Immunohistochemical staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 2. CX3CL1 expression by tumor cells does not correlate with CD3+ or CD68+ infiltrating cells at the tumor invasive front. (A) Immu- nohistochemical analysis of CD3+ or CD68+ cells in CRC samples (n = 100), at the tumor-invasive margin. (B and C) Linear regression analysis. Correlation between CX3CL1 expression and the percentage of CD3+ (B) or CD68+ (C) cells. CX3CL1 expression does not correlate with lympho- cyte or macrophage infiltration. (D) Immunohistochemistry of CX3CR1 showing that in tumor tissue only few immune-infiltrating cells express CX3CR1, whereas in healthy mucosa the majority of immune cells are CX3CR1 positive. (E) Immunofluorescence of CX3CR1 and CD3 or CD68 in human tumor tissues; arrows mark the few double-positive cells. (F) Mouse model of colitis-associated cancer in CX3CR1gfp/gfp and CX3CR1+/gfp

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: Expressing, Immunohistochemistry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 3. Immunohistochemical analysis of CX3CR1 expression in human CRC and correla- tion of the CX3CL1-CX3CR1 axis with clinical outcome. (A) Representative immunoreactivity of CX3CR1 in normal colonic mucosa, precancerous lesion (adenoma), and colorectal cancer. Original magnification 320. (B) Expression scores of CX3CR1 in all tested samples (normal colonic mucosa, n = 18; adenoma, n = 18; colorectal can- cer, n = 100). CX3CR1 is low in normal tissues and precancerous lesions, and strongly increases in tu- mors. Kruskal–Wallis and Dunn’s posttest, ***p , 0.001. (C) Immunofluorescence analysis showing the coexpression of CX3CL1 (red) and CX3CR1 (green) on tumor cells. The presence of several spots of colocalization is shown in the merge panel and close-up. Original magnification 320. (D and E) Kaplan–Meier curves showing disease-free (D) and disease-specific (E) survival, in stage I–III patients according to CX3CL1-CX3CR1 axis expression. Patients with axis-negative tumors (LigLow-RecLow, black line) have significantly shorter DFS and DSS (p = 0.01 and p = 0.001, respectively), compared with axis-positive tumors (LigHigh-RecHigh, red line).

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: Immunohistochemical staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 4. CX3CL1 and CX3CR1 expression is reduced in lymph node metastasis. (A) Percentage of patients with lymph node metastasis at diagnosis and with metachronous metastasis (during follow- up), according to the expression of the chemokine axis by tumor cells. Patients with axis-negative tumors (LigLow-RecLow) have higher frequency of metastases compared with axis-positive tumors (LigHigh-RecHigh). (B) Immunoreactivity of CX3CL1 and CX3CR1 in lymph node metastases and cor- responding primary tumors. Original magnification 320. (C and D) Expression scores of CX3CL1 (C) and CX3CR1 (D) in lymph node metastases (n = 36) and primary tumors (n = 36). Red bars indicate median value. Expression of both CX3CL1 and CX3CR1 in metastatic cells is significantly re- duced. Mann–Whitney, *p , 0.05, ***p , 0.001. LN, lymph node; MT, metastatic tumor; PT, primary tumors.

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: Expressing, Biomarker Discovery, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 6. Expression of the chemokine axis CX3CL1-CX3CR1 re- duces metastatic cell dissemination in vivo. In vivo spleen–liver mouse model of metastasis. NSG mice were spleen injected with 1:1 mixture of tumor cells expressing CX3CL1 or CX3CR1 or Mock cells (n = 10 mice each group). (A) Mice with axis-negative tumors (Mockcherry-Mockgfp, black line), had higher mortality rate compared with axis-positive tumors (CX3CL1cherry-CX3CR1gfp, red line). Log rank, *p , 0.05. (B) Represen- tative histological pictures of liver metastasis from mice receiving different combinations of CX3CL1cherry- or CX3CR1gfp-expressing tumor cells (upper panels, RKO cells; lower panels, NCI-H630). Original magnifica- tion 310. (C and D) Analysis of the liver metastatic area (mean 6 SE of three experiments with RKO, one with NCI-H630). Mice with axis-nega- tive tumors (black bars) had higher number of metastasis compared with axis-positive tumors (red bars). In (D), NCI-H630 cells constitutively produce low levels of CX3CL1, and exogenous expression of CX3CR1 reconstituted the chemokine axis, resulting in fewer metastases (green bar). One-way ANOVA with Tukey’s posttest, *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: Expressing, In Vivo, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 7. FRET analysis of CX3CL1-CX3CR1 interaction in vivo. (A) Immunofluorescence analysis of RKO (left panel) and NCI-H630 (right panel) tu- mors injected in mouse spleens. CX3CL1-CX3CR1 positive-axis tumors show several spots of colocaliza- tion (inset, white asterisk) that are absent in axis-neg- ative tumors. Original magnification 320. (B and C) Phasor plots were used to calculate GFP fluorescence lifetime variation in axis-positive or -negative tumors generated with RKO (B) or NCI-H630 (C) cells: a re- duction in GFP fluorescence lifetime is present only in axis-positive tumors. Green boxes indicate normal GFP lifetime; red boxes indicate the reduced GFP lifetime. (D and E) FRET analysis of CX3CL1-CX3CR1 inter- action. Comparison between mean lifetime of “interacting” versus “noninteracting GFP” in RKO (D) and NCI-H630 cells (E): starting from CX3CL1cherry- CX3CR1gfp–derived phasor plot, the mean lifetime of GFP, in the presence or absence of Cherry (interacting or noninteracting GFP, respectively), was compared. Interacting GFP shows a significantly lower fluo- rescence lifetime in comparison with noninteracting GFP, indicating the presence of FRET events and thus a CX3CL1-CX3CR1 interaction. Student t test, ***p , 0.001.

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: In Vivo, Injection, Generated, Comparison, Derivative Assay

Journal:

Article Title: Chemically synthesized human survivin does not inhibit caspase-3

doi: 10.1110/ps.036145.108

Figure Lengend Snippet: (A) Ligation of (1–45)αCOSR and (46–142) at 1.5 h. The reaction was monitored by analytical HPLC on a Waters XBridge C18 column (4.6 × 150 mm, 3.5 μM) running a 30-min gradient of 25%–45% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. (Insets) Mass spectra of (1–45)αCOSR and (46–142) determined by ESI-MS. (B) Ligated full-length survivin characterized by C18 RP-HPLC and ESI-MS. HPLC conditions: Waters symmetry 300 C18 column (4.6 × 150 mm, 5 μM) running a 30-min gradient of 5%–65% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. (C) CD spectra of synthetic survivin at 5 μM in 5 mM phosphate buffer containing 0.1 mM TCEP, pH 7.5 (thin line), and at 25 μM in 5 mM phosphate buffer containing 0.1 mM TCEP and 50 μM Zn2+, pH 7.5 (thick line). (D) Representative size-exclusion chromatograms of synthetic survivin (3) and molecular mass standards (1, 2, 4, 5). Linear regression analysis of the correlation between logarithmic M r and retention time is illustrated in the inset. (E) Representative raw data from the hydrolysis of Ac-DEVD-AMC by caspase-3 in the absence and presence of different concentrations of XIAP and synthetic survivin. (F) Dose-dependent percent inhibition of caspase-3 by XIAP (filled circles), synthetic survivin without Zn2+ (empty squares), and synthetic survivin in the presence of Zn2+ (filled squares). Each curve is the mean of three independent experiments.

Article Snippet: EnzChek Caspase-3 assay kit #1 was purchased from Invitrogen; recombinant caspase-3 was obtained from Calbiochem and recombinant human XIAP, from R&D Systems.

Techniques: Ligation, Inhibition

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A ) Peptides derived from two known cleavage sites of SARS-CoV-2 spike were designed with C-terminal fluorophore 5-FAM and N-terminal fluorescence resonance energy transfer (FRET) quencher QXL-520. ( B ) FDA-approved and investigational serine protease inhibitors were screened by enzymatic assay to inhibit TMPRSS2 cleavage of SARS-CoV-2 S1/S2 peptide substrate. Relative change in fluorescence with respect to DMSO vehicle is shown. Colors indicate the described target of the drugs screened. All drugs screened at 10 µM final concentration. ( C ) Active form of dabigatran in enzymatic assay for TMPRSS2 inhibition. Relative fluorescence with respect to its corresponding 0.1 N HCl vehicle is shown. ( D ) Schematic of constructs used to generate SARS-CoV-2 spike-pseudotyped/HIV-1-based particles. ( E ) Calu3 cells were treated with 10 µM of the indicated drugs for 24 hr prior to infection with HIV-1 NL /SARS-CoV-2 pseudovirus. Media was changed at 24 hr post infection and pseudoviral entry was measured by nanoluciferase luminescent signal at 40 hr. ( F ) Calu3 cells treated with 10 µM of the indicated drugs were monitored for confluence by Incucyte for 40 hr. ( G ) Pseudoviral entry was measured by nanoluciferase luminescent signal in Calu3 cells treated various concentrations of the indicated drugs for 4 hr prior to infection with SARS-CoV-2 pseudovirus. ( H ) Caco2 cells were infected with lenti-Cas9-blast and U6-sgRNA-EFS-puro-P2A-tRFP and selected. Neutral controls targeting CD4 (not endogenously expressed) or PHGDH intron 1, two sgRNAs each targeting different regions of ACE2 and TMPRSS2 were included. Cells were subsequently infected with HIV-1 NL /SARS-CoV-2 pseudovirus. ( I ) Caco2 cells co-expressing Cas9 and sgRNAs targeting CD4 (not expressed) or TMPRSS2 were treated with 10 µM camostat, nafamostat, or DMSO vehicle. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM. Figure 1—source data 1. Data and summary statistics for enzymatic and pseudovirus assays.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Derivative Assay, Fluorescence, Förster Resonance Energy Transfer, Enzymatic Assay, Concentration Assay, Inhibition, Construct, Infection, Expressing, Two Tailed Test

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A ) TMPRSS2 enzymatic assay was performed in AB1 (20 mM Tris-HCl, pH 7.3, 100 mM NaCl, 1 mM EDTA, fresh 1 mM DTT) or AB2 (50 mM Tris-HCl, 150 mM NaCl, pH 8) using 10 µM of either S1/S2 or S2’ peptide substrate. ( B ) Titration of enzyme concentration was performed (0–1000 nM) with 10 µM S1/S2 substrate. Initial reaction velocity V 0 (rate of change in fluorescent signal) each enzyme concentration with 10 µM S1/S2 peptide substrate.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Enzymatic Assay, Titration, Concentration Assay

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: A549 cells (which do not express ACE2), A549/ACE2 cells (ectopic ACE2 expression from a lentiviral vector), and Caco2 cells (which express endogenous ACE2 and TMPRSS2) infected with HIV-1 NL -based particles pseudotyped with SARS-CoV-2 S or VSV G. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Expressing, Plasmid Preparation, Infection, Two Tailed Test

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A ) Schematic of constructs used to generate SARS-CoV-2 spike-pseudotyped/VSV-based pseudovirus. ( B ) Nanoluciferase luminescent signal following addition of rVSV∆G pseudovirus complemented with VSV G, SARS-CoV-2 S, SARS-CoV S, or without complementation with any envelope protein to Calu3 cells. Each pseudovirus was titrated by adding the indicated volume of inoculum, supplemented with fresh media up to 200 µl/well in a 96-well plate. ( C–F ) Nanoluciferase luminescent signal following infection of ( C ) Caco2, ( D ) Calu3, ( E ) A549/ACE2, or ( F ) Vero cells with rVSV∆G/SARS-CoV-2 pseudovirus pretreated for 4 hr with 10 µM camostat, nafamostat, dabigatran, or otamixaban, compared with uninfected or infected/untreated cells. Expression status of ACE2 and TMPRSS2 for each cell line is indicated. N = 3. Data represented as mean ± SEM.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Construct, Infection, Expressing

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A ) Constructs used for CRISPR experiments. ( B ) Percentage of reads exhibiting wild type, frameshift, or in-frame indels at each locus for the indicated sgRNAs. ( C–F ) Distribution of reads with deletion or insertion by position within amplicon. ( G–J ) Distribution of the size of insertions and deletions in each amplicon. Two sgRNAs targeting ACE2 (g1 and g2) and two sgRNAs targeting TMPRSS2 (g1 and g2) were analyzed.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Construct, CRISPR, Amplification

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: Initial velocities for the cleavage of SARS-CoV-2 spike S1/S2 and S2’ peptide substrates by ( A ) TMPRSS2, ( B ) factor Xa, and ( C ) thrombin were measured over a range of 0–160 µM substrate. From initial velocity values, enzyme kinetic constants ( D ) turnover rate K cat (s –1 ), ( E ) affinity constant K m , and ( F ) specificity constant (K cat /K m ) were obtained for the indicated enzymes with S1/S2 and S2’ peptides. ( G–I ) Heatmaps depict the initial velocity V 0 of cleavage of the indicated peptide substrates and concentrations by ( G ) TMPRSS2, ( H ) factor Xa, and ( I ) thrombin. Figure 2—source data 1. Data and summary statistics for enzymatic assays.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques:

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: Kinetics of SARS-CoV-2 spike peptide substrate cleavage. Kinetic constants obtained from initial velocity studies with varying concentrations of SARS-CoV-2 spike S1/S2 and S2’ peptide substrates. Each estimate is based on seven different concentrations of substrate in 1:2 serial dilution (0–160 µM).

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Serial Dilution

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A–C ) Initial reaction velocity with respect to enzyme concentration for peptide substrates of the SARS-CoV-2 spike S1/S2 site (S1S2), with P1 arginine substituted with alanine (S1S2-P1A), or with substitutions in the P3 and P4 position (RR > SQ) with ( A ) TMPRSS2, ( B ) factor Xa, or ( C ) thrombin. ( D ) List of peptide substrates used in this study. ( E ) Initial reaction velocity of factor Xa cleavage of SARS-CoV-2 S1/S2 or thrombin-R271 peptide substrates in the presence of 0–100 µM phosphatidylcholine/phosphatidylserine (PC/PS) phospholipid vesicles. ( F ) Dilute Russell’s viper venom clotting time (dRVVT) assay of pooled normal human plasma, supplemented with 0–100 µM PC/PS phospholipid vesicles. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Concentration Assay, Coagulation, Clinical Proteomics, Two Tailed Test

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A ) Calu3 cells were infected with rVSV∆G/SARS-CoV-2 pseudovirus with concomitant treatment with vehicle, 250 nM factor Xa, or 250 nM thrombin. Quantification of the ratio of green fluorescent area to total confluence (4 fields/replicate well, 4 wells/condition). ( B ) Nanoluciferase luminescent signal was measured following infection with rVSV∆G/SARS-CoV-2 pseudovirus and the addition of either vehicle, factor Xa, or thrombin. The effect of factor Xa on rVSV∆G complemented with either ( C ) SARS-CoV spike or ( D ) VSV-G was measured by luminescent signal. Luminescent signal was measured following HIV-1 NL /SARS-CoV-2 pseudovirus infection and concomitant treatment with 125–250 nM factor Xa in ( E ) Calu3 cells, ( F ) A549/ACE2, and ( G ) Vero cells following transduction with lentiviral vectors to express GFP or TMPRSS2. Following selection, cells were infected with HIV-1 NL /SARS-CoV-2 pseudovirus and concomitantly treated with 125–250 nM factor Xa. Subsequently, nanoluciferase luminescent signal was determined and plotted relative to vehicle-treated control. *p < 0.05, two-tailed t-test. Data represented as mean ± SEM. Figure 3—source data 1. Data and summary statistics for pseudovirus assays with exogenous proteases.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Infection, Transduction, Selection, Control, Two Tailed Test

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: Initial velocities for the cleavage of 10 µM SARS-CoV-2 spike S1/S2 (top) and S2’ (bottom) peptide substrates by ( A ) TMPRSS2, ( B ) TMPRSS11D/human airway trypsin-like protease ( C ) factor Xa, and ( D ) thrombin were measured in the presence of DMSO vehicle, or 10 µM camostat, nafamostat, otamixaban, or dabigatran. The relative activity of ( E ) factor Xa and ( F ) thrombin were determined over a range of 0–10 µM of the indicated drugs. Calu3 cells were treated with a range of concentrations of nafamostat with or without addition of 250 nM exogenous factor Xa and infected with ( G ) rVSV∆G/SARS-CoV-2 pseudovirus or ( H ) HIV-1 NL /SARS-CoV-2 pseudovirus and infectivity was measured by luminescence. N = 3, data represented as mean ± SEM. Figure 4—source data 1. Data and summary statistics for enzymatic assays to determine the effects of protease inhibitors on host proteases.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Activity Assay, Infection

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet:

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Recombinant, Lysis, Luciferase, Clinical Proteomics, Sequencing, Software