Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: ( A ) sCD13 showed significant correlations with Cit-H3 ( n = 172) and MPO-DNA ( n = 172) in patients with COVID-19 while S100 A8/A9 ( n = 172) showed a positive slope but not a statistically significant correlation. ( B ) Representative images of neutrophils isolated from peripheral blood and analyzed after stimulation with PBS or sCD13. Panels show merged images of NETs in which neutrophil elastase was stained green by immunofluorescence and DNA was stained blue by Hoechst 33342. n = 3 technical replicates. Scale bar: 100 μm. ( C ) sCD13 blocked the staining of PAR4-5F10 antibodies, which recognize only the inactivated/uncleaved form of PAR4 yet had no effect on PAR4-FITC antibodies, which recognize both the activated and inactivated forms. n = 3 technical replicates, repeated 2 times. Original magnification, ×1,000. ( D ) sCD13-induced NETosis was blocked by B1R inhibitor SSR-240612 and PAR4 inhibitor BMS-986120 (all n = 3). ( E ) PMA-induced NETosis was not impacted by the B1R or PAR4 inhibitors (all n = 3). Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Significance was determined by Spearman’s test ( A ) and 1-way ANOVA ( D and E ).

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: Isolation, Staining, Immunofluorescence

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: Single-cell RNA-Seq results of nasopharyngeal/pharyngeal swabs, bronchial brushings and bronchial lavages were generated from patients with COVID-19 ( n = 19) and healthy controls ( n = 5). Data are extracted from Chua et al. . ( A ) Both ANPEP (codes for CD13) and MMP14 are expressed on various epithelial cells and macrophages. ANPEP is also expressed on neutrophils while MMP14 is expressed on mast cells. BDKRB1 (codes for B1R) is expressed on some epithelial cells with the highest expression on secretory cells and ciliated cells. F2RL3 (codes for PAR4) was barely detected in this dataset but seemed to be expressed by epithelial cells. Cell abbreviations are defined in . ( B ) Cellular ANPEP expression in nasopharyngeal cells is elevated in patients with COVID-19 (moderate COVID-19 n = 8, severe COVID-19 n = 11) compared with healthy controls ( n = 5). ( C ) Using the median expression levels of ANPEP in patients, patients with COVID-19 were divided into 2 populations: ANPEP -high and ANPEP -low. Differentially expressed genes in neutrophils from these 2 groups were shown in the volcano plot ( P < 1 × 10 –20 , |log 2 (fold-change)| < 0.25). ( D ) Pathway analysis of the differentially expressed genes in ANPEP -high and ANPEP -low neutrophils showed pathways related to neutrophil degranulation, leukocyte activation, and inflammation. ( E ) The neutrophils from ANPEP -high patients with COVID-19 ( n = 10) showed a gene signature of immature-like neutrophils characterized by the overexpression of genes coding for several granule-content proteins (healthy controls n = 5, ANPEP -low n = 9). ( F ) The score for neutrophil immaturity was higher in critically ill patients with COVID-19 ( n = 11) compared with moderate patients (left, n = 8). The median neutrophil-immaturity score of neutrophils in ANPEP -high patients ( n = 10) was lower than that in ANPEP -low patients (right, n = 9). ( G ) The neutrophil-immaturity signature was most prominent in ANPEP -high patients ( n = 10) as developing neutrophils versus mature neutrophils from the peripheral blood were examined (healthy controls n = 5, ANPEP -low n = 9). Results are expressed as mean ± SD. **** P < 0.0001. Significance was determined by 1-way ANOVA ( B and G ) and Mann-Whitney test ( F ).

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: RNA Sequencing, Generated, Expressing, Activation Assay, Over Expression, MANN-WHITNEY

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: ( A ) Gating scheme of flow cytometry analysis on monocytes and neutrophils. ( B ) CD13, B1R, and PAR4 were expressed on monocytes and neutrophils, while MMP14 showed minimal expression on these cells ( n = 3). ( C ) Histograms showing the changes in CD13, PAR4, and B1R expression in neutrophils and monocytes with or without IL-1β, TNF-α, or IL-6 stimulation. ( D ) Quantification of relative expression of CD13, PAR4, and B1R on neutrophils and monocytes after stimulation with IL-1β, TNF-α, or IL-6 compared with unstimulated control from 3 healthy donors. Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. Significance was determined by 1-way ANOVA. FMO, fluorescence minus 1 (gating control); NT, not treated.

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: Flow Cytometry, Expressing, Control, Fluorescence

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: ( A ) Reanalysis of single-cell RNA-Seq results generated from circulating human neutrophils revealed that immature neutrophils have higher ANPEP expression. Dot plot of ANPEP , IFN, and neutrophil maturity genes for each neutrophil cluster, showing the average expression level and the percentage of cells expressing the gene in each cluster. ( B ) Gating scheme of flow cytometry analysis on mature and immature neutrophils isolated from whole blood. CD10 + CD16 hi defines mature neutrophils while CD10 – CD16 lo defines immature neutrophils. ( C ) Significantly higher expression of CD13 and lower expression of PAR4 were observed in mature neutrophils compared with immature neutrophils while B1R expression showed similar levels. Data generated from 5–10 healthy controls. Results are expressed as mean ± SD. ** P < 0.01. Significance was determined by Wilcoxon’s test.

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: RNA Sequencing, Generated, Expressing, Flow Cytometry, Isolation

Journal: Frontiers in Pharmacology

Article Title: Role of albumin in regulating platelet function

doi: 10.3389/fphar.2026.1734694

Figure Lengend Snippet: Hypoproteinemia was found to promote platelet activation. (A–E) Wash platelet aggregation in WT, ADR and ADR Albumin mice induced by thrombin (A) , U46619 (B) , PAR4-AP (C) , collagen (D) and ADP (E) (n = 5 independent experimental animals). (F–H) Thrombin (F) , U46619 (G) and PAR4-AP (H) induced integrin αIIbβ3 activation in washed platelets from mice (n = 5 independent experimental animals). (I–K) Thrombin (I) , U46619 (J) and PAR4-AP (K) induced P-selectin release from washed platelets from mice (n = 5 independent experimental animals). (L–O) ATP release from washed platelets in mice was induced by thrombin (L) , U46619 (M) , PAR4-AP (N) and collagen (O) (n = 5 independent experimental animals). (P–R) Wash platelet spreading in mice induced by U46619 (P) , mean spreading area of individual platelets (Q) , and proportion of spreading platelets to total platelets in the field of view (R) (n = 3 independent experimental animals). Differences between groups were assessed by one-way ANOVA followed by Dunnett’s post hoc test. Statistics are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Rabbit antibodies against phospho-PKC substrate (2261), phospho-Akt (Ser473) (11E7), Akt (pan) (11E7), PKC alpha (2056), GAPDH (5174), cell lysate buffer (9803), and PAR4-AP (2328) were sourced from Cell Signaling Technology, Inc. (Beverly, MA, United States).

Techniques: Activation Assay

Journal: Frontiers in Oncology

Article Title: The protease-activated receptors are expressed in glioblastoma and differentially modulate adherent versus stem-like growth of LN-18 GBM cells

doi: 10.3389/fonc.2025.1582996

Figure Lengend Snippet: PAR1–4 mRNA expression in GBM specimen and its association with patients´ overall survival time. (A) Comparison of PAR1, PAR2, PAR3 and PAR4 mRNA expression in non-malignant brain (NMB, n=7) and all analyzed GBM samples (both primary and relapsed GBM, n=118). (B) Subdivision of GBM specimen in primary GBM (prGBM, n=78), first (1 st , n=33) and second (2 nd , n=7) relapses and comparison with NMB. (A+B) Gene expression was measured by qPCR. Each mRNA level of the target genes (PAR1-4) was normalized to the mean of GAPDH and β-actin using the 2 -ΔΔct method. Data are shown as scatter plots representing the median as horizontal bars. Mann Whitney U test, *p < 0.05 and ***p < 0.001 for (A) and OneWay ANOVA/Kruskal Wallis test with Dunn’s Multiple Comparison Test, *p < 0.05, **p < 0.01 and *** p < 0.001 for (B) . (C) Kaplan Meier survival analyses of PAR1–4 mRNA expression in GBM patients. Association of the relative mRNA expression of each single PAR receptor with the survival time of patients with primary GBM. The patients were divided into two subgroups depending on the median gene expression. No significant association was found.

Article Snippet: As specific PAR antagonists the following compounds were used: PAR1 antagonist RWJ, PAR2 antagonist FSLLRY-NH2, and PAR4 antagonist tcY-NH2 (all from Tocris Bioscience).

Techniques: Expressing, Comparison, Gene Expression, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: The protease-activated receptors are expressed in glioblastoma and differentially modulate adherent versus stem-like growth of LN-18 GBM cells

doi: 10.3389/fonc.2025.1582996

Figure Lengend Snippet: Impact of PAR subtype specific inhibitors and agonists on viability of LN-18 GBM cells. Adherent and neurospheric LN-18 cells of passage 1 or 3 were treated for 48 or 72h with the respective compounds followed by measurement of cell viability using the Resazurin assay. (A+B) Treatment of adherent (A) and neurospheric (B) LN-18 cells with inhibitors of PAR1 (RWJ), PAR2 (FSLLRY) and PAR4 (tcY) (each 5 and 20 µM), n=3-4. (C+D) Incubation of adherent (C) and neurospheric (D) LN-18 cells with Thrombin (TB, 30 U/ml), Factor Xa (FXa, 30 nM) or PAR subtype specific agonists (AP1 to AP4, each 100 and 200 µM), n=3-4. OneWay ANOVA Dunnett’s Multiple Comparison Test, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. Con.

Article Snippet: As specific PAR antagonists the following compounds were used: PAR1 antagonist RWJ, PAR2 antagonist FSLLRY-NH2, and PAR4 antagonist tcY-NH2 (all from Tocris Bioscience).

Techniques: Resazurin Assay, Incubation, Comparison

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity

doi: 10.1016/j.jtha.2024.03.004

Figure Lengend Snippet: Introduction of the C>T substitution in F2rl3 locus. (A) Sequence alignment of human PAR4 and mouse PAR4. P310 at ECL3 (highlighted in blue) in humans is homologous to P322 in mice. (B) gRNAs were targeted against the region of the mouse F2rl3 locus highlighted in red, while the red letters indicate the substitution target site. (C) Sanger sequencing shows the F2rl3 locus around the gRNA target site of a wild-type mouse (top) and a PAR4-P322L homozygous mouse (bottom) in which both alleles contained C>T substitutions. (D) Protein levels of PAR4 were compared across genotypes using an antibody specific for the mouse protein. (E) The total and surface expression levels of mouse PAR4 WT and P322L on HEK293 Flp-In T-REx stable cell lines were compared by flow cytometry using a V5-FITC-conjugated antibody ( n = 3). FITC, fluorescein isothiocyanate; WT, wild type.

Article Snippet: Two small-molecule PAR4 inhibitors from Bristol Myers Squibb, BMS-986120 and BMS-986141, were the subject of clinical trials and proved to be efficient in preventing cardiovascular events with a good safety profile [ , , ].

Techniques: Sequencing, Expressing, Stable Transfection, Flow Cytometry

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity

doi: 10.1016/j.jtha.2024.03.004

Figure Lengend Snippet: Platelets from PAR4-P322L mice were less responsive to PAR4 agonists. PRP from PAR4 P/P , PAR4 P/L , and PAR4 L/L littermates were stimulated with 50 to 1600 μM PAR4-AP, AYPGKF-NH 2 . (A, B). Gel-filtered platelets from PAR4 P/P , PAR4 P/L , and PAR4 L/L littermates were stimulated with 0.1 to 30 nM thrombin (C, D). The α-granule secretion (A, C) and integrin αIIbβ3 activation (B, D) were measured by flow cytometry using antibodies specific for P-selectin and activated αIIbβ3. Data are presented as means ± SD from 5 independent experiments at each concentration for panels A and B. Data are means from 3 independent experiments at each concentration for panels C and D. PAR4, protease-activated receptor 4; PRP, platelet-rich plasma.

Article Snippet: Two small-molecule PAR4 inhibitors from Bristol Myers Squibb, BMS-986120 and BMS-986141, were the subject of clinical trials and proved to be efficient in preventing cardiovascular events with a good safety profile [ , , ].

Techniques: Activation Assay, Flow Cytometry, Concentration Assay, Clinical Proteomics

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity

doi: 10.1016/j.jtha.2024.03.004

Figure Lengend Snippet: Platelet activation determined by P-selectin-positive or integrin αIIbβ3 activation using flow cytometry.

Article Snippet: Two small-molecule PAR4 inhibitors from Bristol Myers Squibb, BMS-986120 and BMS-986141, were the subject of clinical trials and proved to be efficient in preventing cardiovascular events with a good safety profile [ , , ].

Techniques: Activation Assay, Flow Cytometry

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity

doi: 10.1016/j.jtha.2024.03.004

Figure Lengend Snippet: Reactivity to non-PAR4 agonists was unaffected in platelets from PAR4-P322L mice. PRP from PAR4 P/P , PAR4 P/L , and PAR4 L/L littermates was stimulated with 2.5 to 20 μM ADP (A, B) or 5 nM or 20 nM convulxin (C, D). The α-granule secretion (A, C) and integrin αIIbβ3 activation (B, D) were measured by flow cytometry using P-selectin and activated αIIbβ3 specific antibodies. Data are presented as means ± SD from 5 independent experiments at each concentration. Dots represent individual mice. PAR4, protease-activated receptor 4; PRP, platelet-rich plasma.

Article Snippet: Two small-molecule PAR4 inhibitors from Bristol Myers Squibb, BMS-986120 and BMS-986141, were the subject of clinical trials and proved to be efficient in preventing cardiovascular events with a good safety profile [ , , ].

Techniques: Activation Assay, Flow Cytometry, Concentration Assay, Clinical Proteomics

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity

doi: 10.1016/j.jtha.2024.03.004

Figure Lengend Snippet: Aggregation of platelets from P322L mice had a reduced response to thrombin. Representative tracing of thrombin-mediated platelet aggregation (A-C). Gel-filtered platelets from PAR4 P/P , PAR4 P/L , and PAR4 L/L littermates were stimulated with 0.5 nM (A), 1 nM (B), and 10 nM (C) thrombin. (D) Maximum aggregation of gel-filtered platelets from PAR4 P/P , PAR4 P/L , and PAR4 L/L was compared in response to 0.5 to 10 nM thrombin stimulation. (E) Area under curve of PAR4 P/P , PAR4 P/L , and PAR4 L/L platelet aggregation was compared in response to 0.5 to 10 nM thrombin stimulation. (F) The aggregation rate of PAR4 P/P , PAR4 P/L , and PAR4 L/L platelet aggregation was compared in response to 0.5 to 10 nM thrombin stimulation. Data are representative of 3 independent experiments. Dots represent individual mice. PAR4, protease-activated receptor 4.

Article Snippet: Two small-molecule PAR4 inhibitors from Bristol Myers Squibb, BMS-986120 and BMS-986141, were the subject of clinical trials and proved to be efficient in preventing cardiovascular events with a good safety profile [ , , ].

Techniques:

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity

doi: 10.1016/j.jtha.2024.03.004

Figure Lengend Snippet: PAR4-P322L mice had extended tail bleeding time. Tail bleeding assay was used to evaluate the impact of the PAR4-P322L on hemostatic function. (A) Initial bleeding time was defined as the first time observing the stop of the bleeding regardless of any rebleeding. (B) Total bleeding time was defined as the sum of bleeding times of all bleeding on/off cycles until a stable cessation occurred (no bleeding for 60 seconds). The experiment was terminated at 10 minutes. The data were presented as the percentage of mice that were still bleeding at a specified time point. PAR4, protease-activated receptor 4.

Article Snippet: Two small-molecule PAR4 inhibitors from Bristol Myers Squibb, BMS-986120 and BMS-986141, were the subject of clinical trials and proved to be efficient in preventing cardiovascular events with a good safety profile [ , , ].

Techniques:

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity

doi: 10.1016/j.jtha.2024.03.004

Figure Lengend Snippet: PAR4-P322L mice have longer arterial occlusion times. Arterial thrombosis in our mice was assessed with the ferric chloride–induced carotid artery injury model. (A) The time to occlusion was visually determined as the moment blood flow stopped. The time to complete occlusion was determined in males (B) and females (C). Rhodamine 6G was used to label white blood cells and platelets in real-time over 30 minutes. Representative images are shown (D). PAR4, protease-activated receptor 4.

Article Snippet: Two small-molecule PAR4 inhibitors from Bristol Myers Squibb, BMS-986120 and BMS-986141, were the subject of clinical trials and proved to be efficient in preventing cardiovascular events with a good safety profile [ , , ].

Techniques: