antibodies for par4  (Alomone Labs)

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: