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: 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

Journal: JCI Insight

Article Title: COVID-19 and RA share an SPP1 myeloid pathway that drives PD-L1 + neutrophils and CD14 + monocytes

doi: 10.1172/jci.insight.147413

Figure Lengend Snippet: ( A ) Venn diagram illustrating numbers of unique and shared marker genes of ST TREM2 hi and BALF FABP4 + macrophage clusters as described in . Marker genes were identified prior to integration of data sets ( , ) and were calculated using MAST, setting a minimum percentage of cells in clusters expressing each marker to 40%. Genes considered differentially expressed at P < 0.05 after Bonferroni correction. ( B ) Heatmap illustrating scaled, pseudobulk expression of shared upregulated marker genes from ST and BALF clusters indicated in A . ( C ) Split UMAP plots comparing BALF macrophage clusters in health, and in mild and severe COVID-19, illustrating changes in expression of the TAM receptors AXL and MerTK , with their respective preferred ligands GAS6 and PROS1 . Intensity of purple indicates expression level. ( D ) Heatmap illustrating scaled, pseudobulk expression of TAM receptors and associated ligands by each BALF cluster, across patient groups. TAM receptors and their ligands were significantly differentially expressed in severe COVID-19 versus healthy tissues ( P ≤ 0.005), with Bonferroni correction for multiple comparison, as confirmed by MAST.

Article Snippet: Blood samples were centrifuged (600 g /15 minutes) and plasma aliquots were stored at –80°C until analysis by ELISA for SPP1 (BMS2066; Thermo Fisher Scientific), S100A12 (DY1052; R&D Systems), GAS6 (DY885B; R&D Systems), and PROS1 (NBP2-60585; NOVUS Biological).

Techniques: Marker, Expressing, Comparison

Journal: JCI Insight

Article Title: COVID-19 and RA share an SPP1 myeloid pathway that drives PD-L1 + neutrophils and CD14 + monocytes

doi: 10.1172/jci.insight.147413

Figure Lengend Snippet: ( A ) Patients and healthy donors, shown as the following: n = 121 patients with acute pneumonia ( n = 29 community acquired SARS-CoV-2 – pneumonia, n = 29 mild/moderate COVID-19, n = 63 severe COVID-19), convalescent COVID-19 ( n = 41), and healthy controls ( n = 10). Representative images of lung CT scans. ( B ) Plasma levels of SPP1, S100A12, GAS6, and PROS1 in groups as in A . ( C ) Spearman’s rank correlations between SPP1, S100A12, GAS6, and PROS1 plasma levels in patients with acute COVID-19 pneumonia ( n = 92) with demographic and clinical parameters. Each box displays the r value, and an asterisk indicates statistical significance of P < 0.05. ( D ) Plasma levels of SPP1, S100A12, GAS6, and PROS1 in patients with acute COVID-19 pneumonia ( n = 92) stratified based on lung functions measured by PaO 2 /FiO 2 at the time of hospital admission. Severe respiratory failure was defined by PaO 2 /FiO 2 ≤ 200. ( E ) Percentage of acute COVID-19 pneumonia patients ( n = 92) with PaO 2 /FiO 2 ≤ 200 based on high plasma levels of SPP1 (≥108 ng/mL), S100A12 (≥59 ng/mL), GAS6 (≥24 ng/mL), and PROS1 (≥15 μg/mL). ( F ) COVID-19 patient plasma levels of SPP1, S100A12, GAS6, and PROS1 at the time of hospital admission ( n = 92) stratified based on a patient’s subsequent need to be transferred to ICU. ( G ) Percentage of patients with acute COVID-19 pneumonia ( n = 92) transferred to ICU during the hospitalization based on having high levels of SPP1 (≥108 ng/mL), S100A12 (≥59 ng/mL), GAS6 (≥24 ng/mL), and PROS1 (≥15 μg/mL) at the time of hospital admission. ( B , D , and F ) Data are presented as violin plots with median and interquartile range. Asterisk indicates 1-way ANOVA (Kruskal-Wallis test) with Dunn’s correction for multiple comparisons if more than 2 groups were compared ( B ), or 2-sided Mann-Whitney U was used when 2 groups were compared ( B and D – G ). ( H ) Kaplan-Meier analysis of the rate of transfer of COVID-19 patients to ICU based on their cut-off values for SPP1, S100A12, GAS6, and PROS1 at the time of hospital admission.

Article Snippet: Blood samples were centrifuged (600 g /15 minutes) and plasma aliquots were stored at –80°C until analysis by ELISA for SPP1 (BMS2066; Thermo Fisher Scientific), S100A12 (DY1052; R&D Systems), GAS6 (DY885B; R&D Systems), and PROS1 (NBP2-60585; NOVUS Biological).

Techniques: Clinical Proteomics, MANN-WHITNEY

Journal: JCI Insight

Article Title: COVID-19 and RA share an SPP1 myeloid pathway that drives PD-L1 + neutrophils and CD14 + monocytes

doi: 10.1172/jci.insight.147413

Figure Lengend Snippet: ( A ) Representative images of lung CT scans (transversal and sagittal view) of a COVID-19 patient taken during acute pneumonia and during convalescence (68.60 ± 4.36 days after hospital discharge). ( B ) Plasma levels of SPP1, S100A12, GAS6, and PROS1 in paired plasma samples from COVID-19 patients at the time of acute pneumonia and at the convalescent phase ( n = 26). ( C ) Plasma levels of SPP1, S100A12, GAS6, and PROS1 in convalescent COVID-19 patients ( n = 41) stratified based on the severity of prior acute pneumonia and compared with the levels of healthy donors ( n = 10). ( D ) Plasma levels of IL-6 in acute pneumonias and post–COVID-19. ( E ) SPP1, S100A12, GAS6, and PROS1 in convalescent COVID-19 patients ( n = 41) stratified based on suffering ( n = 36) or not ( n = 5) at least 1 of the symptoms (fatigue, musculoskeletal, or respiratory symptoms). ( B ) Data are presented as before-and-after plot. Wilcoxon test on paired samples was used, and exact P values are provided on the graphs. ( C – E ) Data are presented as violin plots with median and interquartile range. Asterisks indicate 1-way ANOVA with correction for multiple comparisons if more than 2 groups were compared, or 2-sided Mann-Whitney U test was used when 2 groups were compared ( C – E ). Exact P values are provided on the graphs.

Article Snippet: Blood samples were centrifuged (600 g /15 minutes) and plasma aliquots were stored at –80°C until analysis by ELISA for SPP1 (BMS2066; Thermo Fisher Scientific), S100A12 (DY1052; R&D Systems), GAS6 (DY885B; R&D Systems), and PROS1 (NBP2-60585; NOVUS Biological).

Techniques: Clinical Proteomics, MANN-WHITNEY