Journal: Blood

Article Title: Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus

doi: 10.1182/blood.2019003630

Figure Lengend Snippet: Generation of a mouse model lacking PS expression in platelets (Pros1lox/loxPf4-Cre+). (A) Genotyping results of mice with normal PS expression (Pros1lox/loxPf4-Cre−), Pros1lox/−, and Pros1lox/loxPf4-Cre+ mice using genomic DNA extracted from ear tissues. The presence of lox sequences in Pros1 alleles and of Pf4-Cre transgene was evaluated by 2 independent multiplex PCRs. PCR products were then subjected to electrophoresis. The lox band had a lower molecular weight (338 bp) compared with the null band (570 bp), in accordance with Saller et al25; the Pf4-Cre+ band has a higher molecular weight (450 bp) in comparison with the internal PCR control (324 bp). (B) PS antigen levels in platelet lysates from Pros1lox/loxPf4-Cre−, Pros1lox/loxPf4-Cre+, and Pros1lox/− mice. Results are expressed as mean ± standard error of the mean (SEM) of percentage relative to the pooled normal platelet lysate. **P ≤ .01; ****P ≤ .0001. (C) PS antigen levels in plasma samples from Pros1lox/loxPf4-Cre−, Pros1lox/loxPf4-Cre+, and Pros1lox/− mice. Results are expressed as mean ± SEM of percentage relative to the pooled normal plasma. ns, not significant; ****P ≤ .0001. (D-E) PS isoforms in plasma and platelet after activation by thrombin. Western blotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions was performed using a monoclonal antibody raised against murine PS (D) and a polyclonal antibody against human PS (E). P, platelet; S, platelet releasate; Thr, thrombin. (F) Confocal microscopy of resting and thrombin-activated mouse Pros1lox/loxPf4-Cre− and Pros1lox/loxPf4-Cre+ platelets, stained for -tubulin (in magenta), CD62p (in green), and PS (in red). Scale bar, 2 μm. Acquired z stacks were used for volumes and surface rendering by Imaris software.

Article Snippet: In addition, the clot elasticity attributable to platelets (MCE plt ) 39 was comparable in Pros1 lox/lox Pf4-Cre − mice and Pros1 lox/lox Pf4-Cre + mice (211 ± 10 vs 209 ± 62, respectively; P > .05). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 Lack of PS in platelets affects global hemostasis assessment ex vivo and in vivo. (A-B) Whole blood activation of coagulation and clot polymerization determined by ROTEM analysis rotational (thromboelastography). (A) Extrinsic‐activated rotational thromboelastometry (EXTEM) assay, (B) extrinsically activated thromboelastometric test with cytochalasin D (FIBTEM).

Techniques: Expressing, Multiplex Assay, Electrophoresis, Molecular Weight, Comparison, Control, Clinical Proteomics, Activation Assay, Western Blot, Polyacrylamide Gel Electrophoresis, Confocal Microscopy, Staining, Software

Journal: Blood

Article Title: Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus

doi: 10.1182/blood.2019003630

Figure Lengend Snippet: Lack of PS in platelets does not affect platelet activation state in steady state, platelet aggregation ex vivo, and platelet-dependent VTE in vivo. (A) PF4 antigen levels in plasma from Pros1lox/loxPf4-Cre− and Pros1lox/loxPf4-Cre+ mice (ns, not significant; P = .145). Data are expressed as median. The normal range of the PF4 antigen concentration in mouse plasma (0.26-4.70 µg/mL) provided by the manufacturer is indicated in the y-axis by dashed lines. (B) Flow cytometry analysis of CD62p and CD41 on resting and convulxin (10 µg/mL)-activated or thrombin (10 U/mL)-activated platelets. (C) Platelet Function Analyzer-100 closure time using collagen-ADP and collagen-epinephrine (collagen-Epi) cartridges to activate platelets in whole blood from Pros1lox/loxPf4-Cre− and Pros1lox/loxPf4-Cre+ (n = 5/genotype). Data are expressed as mean ± SEM. (D) Aggregation of washed platelets suspensions were initiated by adding different concentrations of collagen, ADP (in presence of 2 mg/mL fibrinogen), and thrombin. Results are given as mean ± SEM of at least 3 independent experiments; each of them was obtained from platelet pools (n = 4 mice/genotype). (E) Venous thromboembolism model induced by an intravenous injection of collagen-epinephrine in Pros1lox/loxPf4Cre− (straight line; n = 16) and mice Pros1lox/loxPf4Cre+ (dashed line; n = 13) mice.

Article Snippet: In addition, the clot elasticity attributable to platelets (MCE plt ) 39 was comparable in Pros1 lox/lox Pf4-Cre − mice and Pros1 lox/lox Pf4-Cre + mice (211 ± 10 vs 209 ± 62, respectively; P > .05). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 Lack of PS in platelets affects global hemostasis assessment ex vivo and in vivo. (A-B) Whole blood activation of coagulation and clot polymerization determined by ROTEM analysis rotational (thromboelastography). (A) Extrinsic‐activated rotational thromboelastometry (EXTEM) assay, (B) extrinsically activated thromboelastometric test with cytochalasin D (FIBTEM).

Techniques: Activation Assay, Ex Vivo, In Vivo, Clinical Proteomics, Concentration Assay, Flow Cytometry, Injection

Journal: Blood

Article Title: Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus

doi: 10.1182/blood.2019003630

Figure Lengend Snippet: Absence of PS expression in platelets affects resistance to APC- and TFPI-dependent PS activity. (A-B) APC-dependent activity assessed by thrombin generation in PRP (150 × 109/L platelets). Representative thrombin generation curves in presence of different amount of WT mAPC and L38D mAPC or buffer (no APC) in PRP from Pros1lox/loxPf4-Cre− (A) and Pros1lox/loxPf4-Cre+ mice (B). (C-D) Washed platelet suspension (150 G/L) from Pros1lox/loxPf4-Cre− (C) and Pros1lox/loxPf4-Cre+ mice (D) were reconstituted in platelet-free plasma (PFP) from F8−/−Pros1−/− mice, lacking plasma PS to evaluate the contribution of platelet PS to APC activity. (E-F) To evaluate the contribution of platelet PS to the thrombin potential, thrombin generation was measured in Pros1lox/loxPf4-Cre− and Pros1lox/loxPf4-Cre+ mice. ETP values from PRP (200 G/L platelets) (E) and PFP (F) are shown for both genotypes. (G) ETP change between PRP and PFP [(ETPPRPx100/ETPPFP)-100)] in Pros1lox/loxPf4-Cre− and Pros1lox/loxPf4-Cre+ mice. Data are expressed as mean ± SEM. *P < .05; **P ≤ .01. (H) FXa in activated Pros1lox/loxPf4-Cre− and Pros1lox/loxPf4-Cre+ platelets; representative experiment obtained from a pool of 5 to 6 mice/genotype.

Article Snippet: In addition, the clot elasticity attributable to platelets (MCE plt ) 39 was comparable in Pros1 lox/lox Pf4-Cre − mice and Pros1 lox/lox Pf4-Cre + mice (211 ± 10 vs 209 ± 62, respectively; P > .05). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 Lack of PS in platelets affects global hemostasis assessment ex vivo and in vivo. (A-B) Whole blood activation of coagulation and clot polymerization determined by ROTEM analysis rotational (thromboelastography). (A) Extrinsic‐activated rotational thromboelastometry (EXTEM) assay, (B) extrinsically activated thromboelastometric test with cytochalasin D (FIBTEM).

Techniques: Expressing, Activity Assay, Suspension, Clinical Proteomics

Journal: Frontiers in Cellular Neuroscience

Article Title: Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

doi: 10.3389/fncel.2017.00411

Figure Lengend Snippet: Primers used for qPCR.

Article Snippet: Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

doi: 10.3389/fncel.2017.00411

Figure Lengend Snippet: Primer sequences used for cloning.

Article Snippet: Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C.

Techniques: Cloning, Construct, Sequencing

Journal: Frontiers in Cellular Neuroscience

Article Title: Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

doi: 10.3389/fncel.2017.00411

Figure Lengend Snippet: Volcano plot of all 1240 proteins from the proteomic screen and DAVID analysis of the 80 proteins regulated by eIF2B𝜀 Arg191His identifies a difference in proteins that migrate through the secretory pathway. (A) In the volcano plot the proteins in darker gray have a p -value < 0.05 and are considered significantly differently synthesized between wt and 2b5 ho astrocyte cultures. The p -values are plotted as Log(10) and p < 0.05 corresponds to -Log10 > 1.12. The fold change between wt and 2b5 ho is plotted as Log(2) and a fold change of >2 corresponds to Log(2) > 1. 80 proteins are significantly different between wt and 2b5 ho astrocytes of which 72 are increased and 8 are decreased in 2b5 ho astrocytes. The proteins SLC3A2, PROS1, and GAS6 are highlighted in black. (B) The figure shows the overrepresentation analysis ( p -value < 0.05) between the significantly different proteins with the proteins found in the proteome as a background. The pathway analysis reveals an overrepresentation of proteins with a signal peptide, transmembrane domain, N -linked glycosylation site(s) and/or disulfide bond(s). These terms together pinpoint targeting of proteins to the secretory pathway, some of which are post-translationally modified, as being affected by this mutation.

Article Snippet: Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C.

Techniques: Synthesized, Glycoproteomics, Modification, Mutagenesis

Journal: Frontiers in Cellular Neuroscience

Article Title: Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

doi: 10.3389/fncel.2017.00411

Figure Lengend Snippet: mRNA and protein levels of candidate proteins in astrocyte cultures. (A) mRNA levels of the investigated candidate were investigated with qPCR. The mRNA levels were similar between wt and 2b5 ho astrocytes ( n ≥ 5), with the exception of the Fndc3c1 mRNA, which was increased in 2b5 ho astrocytes. (B) The total protein levels of PROS1 and SLC3A2 were determined by Western blot. The graph shows the mean ± SD. The protein levels were increased in 2b5 ho cultures compared to wt cultures ( n ≥ 3). ∗∗ p < 0.01. Post-translational modifications were overall not affected in 2b5 ho astrocytes. Western blot analyses did not show whether PROS1 in astrocytes was post-translationally modified or not. Staining of all proteins (loading control) is shown in Supplementary Figure .

Article Snippet: Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C.

Techniques: Western Blot, Modification, Staining, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

doi: 10.3389/fncel.2017.00411

Figure Lengend Snippet: In vitro analyses of candidate gene expression regulation do not reveal difference in promoter or 5′-UTR efficiency in wt and 2b5 ho astrocytes. pNL1.1- Scrn1 , - Gas6 and - Pros1 constructs and pNL1.1- Sorcs1 , - Ddr1 , - Gas6 and - Pros1 5′UTR constructs were transfected into wt and 2b5 ho astrocytes. Nanoluciferase activity was measured 3 days post transfection and corrected for firefly luciferase activity expressed from the pGL3- Gapdh internal standard. Every culture is shown as a single dot and bars represent the mean ± SD ( n = 5–9). Replacement of the authentic Pros1 or Gas6 promoter by the Gapdh promoter increased the nanoluciferase expression by approximately twofold. pNL1.1- Gapdh was used as reference non-regulated promoter.

Article Snippet: Membranes were blocked in 5% (w/v) milk powder and stained with the antibody against PROS1 (16910-1-AP, Proteintech) or SLC3A2 (LS-C334231, LSBio) overnight (16 h) at 4°C.

Techniques: In Vitro, Gene Expression, Construct, Transfection, Activity Assay, Luciferase, Expressing

Journal: Frontiers in immunology

Article Title: Identification and validation of key molecules associated with humoral immune modulation in Parkinson's disease based on bioinformatics.

doi: 10.3389/fimmu.2022.948615

Figure Lengend Snippet: FIGURE 7 The expression levels of PPBP, PROS1 and LCN2 from PD patients and healthy controls in GSE100054. (A–C) The expression of PPBP, PROS1 and LCN2 in GSE100054. p < 0.05 was considered to be statistically different.

Article Snippet: The concentrations of PPBP, PROS1, and LCN2 in the plasma of PD and HC were measured using commercially available enzyme-linked immunosorbent assay kits (CUSABIO, Wuhan, China) in accordance with manufacturer’s instructions, which were detected at 450 nm with a microplate reader and calculated by comparing the OD value of the samples to a standard curve.

Techniques: Expressing

Journal: Frontiers in immunology

Article Title: Identification and validation of key molecules associated with humoral immune modulation in Parkinson's disease based on bioinformatics.

doi: 10.3389/fimmu.2022.948615

Figure Lengend Snippet: FIGURE 8 PROS1, PPBP, and LCN2 expression from PD patients and healthy controls. (A) The PROS1 expression in whole blood from PD (n = 16) was higher than HC (n = 16); (B) The PPBP expression in PBMC from PD (n = 22) was lower than HC (n = 16); (C) The LCN2 expression in PBMC from PD (n = 22) was higher than HC (n = 16). HC represents healthy controls, PD represents Parkinson’s disease. * p < 0.05, ** p < 0.01.

Article Snippet: The concentrations of PPBP, PROS1, and LCN2 in the plasma of PD and HC were measured using commercially available enzyme-linked immunosorbent assay kits (CUSABIO, Wuhan, China) in accordance with manufacturer’s instructions, which were detected at 450 nm with a microplate reader and calculated by comparing the OD value of the samples to a standard curve.

Techniques: Expressing

Journal: Frontiers in immunology

Article Title: Identification and validation of key molecules associated with humoral immune modulation in Parkinson's disease based on bioinformatics.

doi: 10.3389/fimmu.2022.948615

Figure Lengend Snippet: FIGURE 9 Plasma levels of PPBP, PROS1 and LCN2 in PD patients (n=69) and healthy controls (n=36). (A) Comparison of the expression levels of plasma PPBP; (B) Comparison of the expression levels of plasma PROS1; (C) Comparison of the expression levels of plasma LCN2. HC represents healthy controls, PD represents Parkinson’s disease. ** P < 0.01, **** P < 0.0001.

Article Snippet: The concentrations of PPBP, PROS1, and LCN2 in the plasma of PD and HC were measured using commercially available enzyme-linked immunosorbent assay kits (CUSABIO, Wuhan, China) in accordance with manufacturer’s instructions, which were detected at 450 nm with a microplate reader and calculated by comparing the OD value of the samples to a standard curve.

Techniques: Clinical Proteomics, Comparison, Expressing

Journal: Comprehensive Physiology

Article Title: Porcine Fetal Hypothyroidism Induces Temporal and Tissue‐Specific Alterations in the Insulin‐Like Growth Factor System

doi: 10.1002/cph4.70028

Figure Lengend Snippet: IGFBP levels in fetal serum. (A) Example Western Ligand Blot showing human recombinant protein standards in the left lane, with the right lane showing the six bands detected in fetal porcine serum. Relative density of (B) a 26 kDa IGFBP, (C) a 28 kDa IGFBP, (D) IGFBP2, (E) the 40 kDa IGFBP3, and (F) IGFBP4 in fetal porcine serum derived from fetuses ( n = 11–12/group) at various gestational timepoints following 21 days of maternal CON or MMI treatment. Relative density was calculated relative to a consistent reference sample run on each gel, with unique letter superscripts denoting statistically significant ( p < 0.05) changes in density in the CON group over time, and statistical differences between the CON and MMI group within each timepoint denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001. Data are presented as the estimated marginal means with 95% confidence intervals, as extracted from a linear model including gestational day, treatment group, and the interaction.

Article Snippet: In addition to samples of interest, each blot contained a lane of pooled recombinant human protein standards representing IGFBPs ‐1, ‐2, ‐3, and ‐4 (R&D Systems), which were used as reference points for identification of bands of interest, as well as a lane of pooled fetal serum which was used to calculate the relative density of each band of interest for analysis.

Techniques: Western Blot, Recombinant, Derivative Assay