Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: The cytometric analysis (SSC and FCS) of isolated human resting platelets (A, D) , gated and labeled PAR-1 without activation (B, E) , and gated and labeled PAR-1 with activation by 10 µM TRAP (C, F) ; labeled with anti-CD61-FITC and PAR-1-APC antibodies (G–I) . The level of PAR-1 expression was read from gates P1. Markers M1 and M2 indicate the gates for microparticles and normal platelets, with the PAR-1 analysis applied to the summed population. An example image from a patient with DM is shown.

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques: Isolation, Labeling, Activation Assay, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: The percentage of PAR-1 receptor expression before and after the addition of the thrombin receptor activating peptide (TRAP) in blood samples from patients with diabetic macroangiopathy (DM), the control group (CONTROL), and atherosclerosis obliterans (AO).

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques: Expressing, Control

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: (A) Separation of DNA molecules in a 3% agarose gel of PAR-1 gene amplification products with the −506 I/D polymorphism. Lanes: 1 – homozygous I/I, 2 – heterozygous I/D, 3 – homozygous D/D, M–GeneRuler™ 50bp DNA Ladder (Fermentas). (B) The percentage distribution of the −506 I/D polymorphism variants in the PAR-1 gene: homozygous D/D (blue), heterozygous I/D (red), and homozygous I/I (green).

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques: Agarose Gel Electrophoresis, Amplification

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: (A) The result of the restriction digestion of PCR products with the MvaI enzyme to check for the presence of the −1426 C/T polymorphism in the PAR-1 gene. Lanes: 1 – 6 homozygotes C/C, M–GeneRuler™ 100bp DNA Ladder (Fermentas). (B) The percentage distribution of the variants of the −1426 C/T polymorphism in the PAR-1 gene: homozygote C/C (blue color), heterozygote C/T (red color), homozygote T/T (green color).

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: (A) Example separations of amplification products of the DNA fragment encompassing the IVSn-14 A/T polymorphism site of the PAR-1 gene using the SNaPshot method. Alleles were determined based on the size of primers and the colors of fluorescently labeled ddNTPs (terminators) incorporated during the primer extension reaction. (A) red peak, wild-type homozygote (AA); (B) green and red peaks, heterozygote (AT); (C) green peak, mutated homozygote (TT). (B) The percentage distribution of the variants of the IVS-14 A/T polymorphism of the PAR-1 gene is as follows: homozygote A/A (blue color), heterozygote A/T (red color), and homozygote T/T (green color).

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques: Amplification, Labeling

Journal: Frontiers in Molecular Biosciences

Article Title: The predictive role of protease-activated receptor (PAR-1) polymorphisms and activated microplatelets on the severity of atherosclerosis – preliminary studies

doi: 10.3389/fmolb.2025.1662954

Figure Lengend Snippet: Multivariate analysis: (A–D) Number of microparticles with PAR-1+TRAP; (E–F) Number of microparticles with BMI (Figure 4.12E-F) and age with smoking.

Article Snippet: Next: For the PAR-1 test without PLT activation, 5 μL of PAR-1-APC antibodies at a concentration of 5 μg/5 × 10 5 cells (Allophycocyanin (APC)-conjugated mouse monoclonal anti-human PAR-1; clone# 731115; mouse isotype: IgG1, R&D Systems, Minneapolis, Canada) and 5 μL of CD61-FITC antibodies (Monoclonal Mouse Anti-Human CD61, Platelet Glycoprotein IIIa/FITC, Clone Y2/51, code: F0803, DakoCytomation, Glostrup, Denmark) were added.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Blockade of PAR2 Signaling by Punicalagin as a Therapeutic Strategy for Atopic Dermatitis

doi: 10.3390/ijms26188920

Figure Lengend Snippet: Potency and selectivity of PCG for PAR2. ( A , B ) Inhibition of PAR2-mediated calcium mobilization by PCG in HaCaT cells. Cells were pretreated with PCG for 10 min before stimulation with 30 µM PAR2-AP or 30 U/mL trypsin (TRY). ( C , D ) Effect of PCG on PAR1-mediated calcium mobilization in HaCaT cells. Cells were pretreated with PCG for 10 min before stimulation with 30 µM PAR1-AP or 30 U/mL thrombin (TRB). ( E ) Summary of dose–response curves for PAR2 and PAR1 inhibition by PCG.

Article Snippet: PAR1 activating peptide (PAR1-AP, TRLLR-NH 2 ) and PAR2 activating peptide (PAR2-AP, SLIGRL-NH 2 ) were synthesized by Cosmogenetech Co., Ltd. (Seoul, Republic of Korea).

Techniques: Inhibition

Journal: bioRxiv

Article Title: Angiogenic CD8 T cells from PWH induce Granzymes-dependent PAR1 activation promoting endothelial inflammation

doi: 10.1101/2025.08.06.668764

Figure Lengend Snippet: In vivo expression of PAR1 in splenic cells from naïve C57BL/6-F2r fl/fl (n= 5) and C57BL/6 (n= 5) mice analyzed by flow cytometry. (A) Cells were analyzed after gating on live cells, and singlets and CD45 + lymphocytes. Representative contour plots gating cell types: NK cells (CD3 - NK1.1 + ), B cells (MHCII + B220 + ), CD4 (CD3 + CD4 + ) and CD8 T cells (CD3 + CD8 + ). (B) Representative histogram of tdTomato (PAR1) expression in CD4 and CD8 T cells, B cells and NK cells from C57BL/6 mice (solid grey) and C57BL/6-F2r fl/fl mice (black line). (C) Median Fluorescence Intensity (MFI) of tdTomato expression of total splenic cells from C57BL/6 mice (SP) and B cells, NK and CD4 and CD8 T cells and NK cells from C57BL/6-F2r fl/fl mice. (D) Relative F2r mRNA expression in sorted B cells and purified by magnetic isolation of CD4 and CD8 T cells from C57BL/6-F2r fl/fl mice (n= 6). (E) C57BL/6-F2r fl/fl mice were infected with LCMVArm strain (2 x 10 5 pfu, i.v). Splenic and lung cells were assessed at day 6 and 21 p.i. by flow cytometry. (F) Representative histogram of tdTomato (tdT) expression in total CD8 T cells from naïve (N) and infected mice at day 6 and 21 p.i. (solid gray). Overlayed, tdTomato expression in LCMVGp33-specific CD8 T cells detected by H2-D b LCMVGp33 pentamer staining (red line). (F) Median Fluorescence Intensity (MFI) of tdTomato expression in LCMVGp33-specific CD8 T cells detected by H2-D b LCMVGp33 pentamer staining. Dashed line represents level expression of total naïve CD8 T cells. (G) Representative plot of lung endothelial cells (EC). After excluding dead cells and doublets, ECs were gated as CD326 - CD45 - CD31 + . Frequency and cell count of lung endothelial cells from naïve (N) and infected mice at days 6 and 21 p.i. Frequencies of ECs are expressed as frequency of total live cells. (H) Median Fluorescence Intensity (MFI) of tdTomato expression in lung ECs from naïve (N) and infected mice (day 6 and day 21p.i.). The bar graph is represented by box and whisker showing the median value with first and third quartiles in the box, with whiskers extending to the minimum and maximum values. The data in line graphs represent mean±SEM in each time point. Data are pooled from three independent experiments with 5-9 naïve animals (C, E and F) and 8-17 per time point (F and H). Statistical analysis was performed using non-parametric Mann-Whitney test. P value < 0.05 was considered significant.

Article Snippet: As the positive control, HUVEC were incubated with 100 μM of PAR1 agonist TFLLR-NH2 (Tocris Bioscience, UK) and control peptide RLLFT-NH2 (Tocris Bioscience, UK).

Techniques: In Vivo, Expressing, Flow Cytometry, Fluorescence, Purification, Isolation, Infection, Staining, Cell Counting, Whisker Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Angiogenic CD8 T cells from PWH induce Granzymes-dependent PAR1 activation promoting endothelial inflammation

doi: 10.1101/2025.08.06.668764

Figure Lengend Snippet: (A) Schematic presentation of CD8 T cell depletion. C57BL/6-F2r fl/fl mice were infected with LCMVArm strain (2 x 10 5 pfu, i.v). At day 15 p.i mice were administered anti-CD8α mAb and IgG2b isotype for control mice. Frequency and cell count of CD8 T cells at day 21p.i in CD8 T cell depleted mice and IgG treated control mice assessed by flow cytometry. (B) Lung endothelial cell frequency and count from CD8 T cell depleted and control mice at day 21 p.i. Frequencies of ECs are expressed as frequency of total live cells. (C) Representative contour plot of CD31 high and CD309 expression by lung CD8 T cells from naïve mice and infected mice. Frequency and cell count of CD31 high CD8 T cells from naïve (N) mice and infected mice at day 6 and 21 p.i. (D) Lung CD31 high and CD31 neg CD8 T cells, representative contour plot of secretion of IFNψ, TNFα secretion and CD107a upon in vitro stimulation with LCMVGp33-44 peptide. DMSO was used as control. (E) Representative contour plot of KLRG1 and CD127 expression by LCMVGp33-specific CD8 T cells. Frequency of KLRG1 - CD127 + (R1), KLRG1 + CD127 + (R2) and KLRG1 + CD127 - and KLRG1 - CD127 - (R3+R4) LCMVGp33 specific CD8 T cells from lung at days 6 and 21 p.i.. (F) Lung CD31 high and CD31 neg bulk CD8 T cells were sorted from naïve mice and infected mice at 21 p.i. and stimulated in vitro with anti-CD3 and anti-CD28 mAbs coated plate for 3 days. Detection of VEGF in the supernatants of the culture. The bar graphs are represented by box and whisker showing the median value with first and third quartiles in the box, with whiskers extending to the minimum and maximum values. The data in line graphs represent mean±SEM in each time point. Data are pooled from two independent experiments with 8 naïve animals (A and B) and 7-16 per time point (C, D and E). Sorted cells from 3 naïve and 6 infected mice per time point (G). Statistical analysis was performed using non-parametric Mann-Whitney test. P value < 0.05 was considered significant.

Article Snippet: As the positive control, HUVEC were incubated with 100 μM of PAR1 agonist TFLLR-NH2 (Tocris Bioscience, UK) and control peptide RLLFT-NH2 (Tocris Bioscience, UK).

Techniques: Infection, Control, Cell Counting, Flow Cytometry, Expressing, In Vitro, Whisker Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Angiogenic CD8 T cells from PWH induce Granzymes-dependent PAR1 activation promoting endothelial inflammation

doi: 10.1101/2025.08.06.668764

Figure Lengend Snippet: Circulating T ang were analyzed in PBMCs from PWH (n= 25, red symbols) and PWoH (n= 17, blue symbols) by flowcytometry. After gating on live cells and singlets, CD4 and CD8 T cells were analyzed based on expression of CD31 and CXCR4: (A) Representative contour plots for gating angiogenic T cells, CD4 T ang (CD31 + CXCR4 + ) and CD8 T ang (CD31 + CXCR4 + ). (B) Frequency of CD4 T ang and CD8 T ang from PWH and PWoH. CD4 T ang and CD8 T ang expression of: ( C) CX3CR1 and PAR1 (D) CD49f and PAR1 (E) granzyme A (GZMA) and B (GZMB). F, G and H. Relationship between expression of CD4 and CD8 T ang cells and atherosclerotic cardiovascular disease (ASCVD) risk score in PWH. (I) Relationship between expression of plasma levels of sVCAM-1, sICAM-1, IL-6 and granzyme A in PWH (n= 25). The bar graph is represented by box and whisker showing the median value with first and third quartiles in the box, with whiskers extending to the minimum and maximum values. Statistical analysis was performed using non-parametric Mann-Whitney test. P value < 0.05 was considered significant. Correlations were performed using non-parametric Spearman correlation and p value ≤ 0.01 was considered significant.

Article Snippet: As the positive control, HUVEC were incubated with 100 μM of PAR1 agonist TFLLR-NH2 (Tocris Bioscience, UK) and control peptide RLLFT-NH2 (Tocris Bioscience, UK).

Techniques: Expressing, Clinical Proteomics, Whisker Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Angiogenic CD8 T cells from PWH induce Granzymes-dependent PAR1 activation promoting endothelial inflammation

doi: 10.1101/2025.08.06.668764

Figure Lengend Snippet: (A) Diagram illustrates the molecular mechanism of cleavage dependent PAR1 activation via serine proteases. Inhibition of PAR1 activation by ATAP-2 mAb and antagonist SCH530348 (vorapaxar). Peptide agonist (mimics the tethered ligand) and cleavage independent PAR1 activation. The figure was generated with Biorender. (B) Image of Granzyme A induced intracellular calcium mobilization in HUVEC. Intracellular calcium mobilization kinetics in HUVEC stimulated with granzyme A and thrombin as control. Each line represents the kinetics overtime of individual cells (lower panel). (C, D and E) HUVEC were stimulated with 100 nM of rh GZMA and GZMK overnight in the presence or absence of 10 μg/mL of ATAP-2 mAb or 300 nM of PAR1 antagonist SCH530348. ( F, G and H ) HUVEC were stimulated with 100 μM of PAR1 agonist peptide (TFLLR-NH2) and peptide control (RLLFT-NH2) as positive and negative controls respectively. IL-6, IL-8/CXCL8 and Angiopoietin-2 were measured in the supernatants of the cultures. The bar graph is represented by box and whisker showing the median value with first and third quartiles in the box, with whiskers extending to the minimum and maximum values. Statistical analysis was performed using non-parametric Mann-Whitney test. P value <0.05 was considered significant.

Article Snippet: As the positive control, HUVEC were incubated with 100 μM of PAR1 agonist TFLLR-NH2 (Tocris Bioscience, UK) and control peptide RLLFT-NH2 (Tocris Bioscience, UK).

Techniques: Activation Assay, Inhibition, Generated, Control, Whisker Assay, MANN-WHITNEY