Journal: JCI Insight

Article Title: Platelets impair the resolution of inflammation in atherosclerotic plaques in insulin-resistant mice after lipid lowering

doi: 10.1172/jci.insight.193593

Figure Lengend Snippet: ( A ) Study design. Eight-week-old Ldlr –/– male mice fed a high-fat, high-cholesterol (HFHC) diet for the whole duration of the study (23 weeks). After the atherosclerosis progression period (obese baseline, Ob), mice were injected with ApoB ASO to lower lipids (lipid-lowering period). All lipid-lowered mice were split into 2 groups, injected either with isotype control antibody (IgG) or platelet depletion antibody (αCD42b) every 3 days for a total of 3 weeks. ( B ) Platelet counts in circulating blood of Ob, IgG, and αCD42b 3 days before harvest. ( C ) Blood glucose measurements (lean n = 16, Ob n = 47) and ( D ) area of curve (AOC) quantification of glucose tolerance tests (GTTs) from lean, Ob, IgG, and αCD42b mice after 16 weeks of HFHC diet during atherosclerosis progression. ( E ) Blood glucose measurements (Ob n = 3, IgG n = 4, αCD42b n = 4) and ( F ) AOC quantification of GTTs from Ob, IgG, and αCD42b mice after 22 weeks of HFHC diet during atherosclerosis resolution. Mean fluorescent intensity (MFI) of platelet ( G ) JonA and ( H ) P-selectin in nonstimulated (NS) samples and upon 100 μM PAR4-AP agonist stimulation of lean and Ob circulating blood samples. ( I ) Mean platelet volume (MPV) from circulating blood of lean, Ob, and IgG-treated mice, 3 days before harvest. In C and E , error bars represent SD. Data were analyzed by Kruskal-Wallis with Dunn’s post hoc test ( B and F ), unpaired Mann-Whitney test ( C , D , and H ), unpaired Student’s t test ( G ), or ordinary 1-way ANOVA with Tukey’s multiple-comparison test ( I ). P values are shown in graphs.

Article Snippet: The whole blood was stained with anti–mouse JonA-PE (M023-2, Emfret Analytics) and anti–mouse P-selectin Alexa Fluor 647 (563674, BD Biosciences) and then treated with PAR4-AP platelet agonist (100 μM; MedChemExpress) for 15 minutes at room temperature.

Techniques: Injection, Control, MANN-WHITNEY, Comparison

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: PAR4 Inhibition Reduces Coronary Artery Atherosclerosis and Myocardial Fibrosis in SR-B1/LDLR Double Knockout Mice

doi: 10.1161/ATVBAHA.123.319767

Figure Lengend Snippet: RAG8 treatment impairs PAR (protease-activated receptor) 4–mediated platelet activation. A , SR-B1 (scavenger receptor class B type I)/LDLR (low-density lipoprotein receptor) double knockout (DKO) mice fed the high-fat, high-cholesterol diet containing cholate (HFCC) for 20 days and treated with the RAG8 or the inactive control SRQ8 pepducins were anesthetized, and their tails were transected across the vein ≈0.5 cm from the tip and placed in a cuvette with warm saline. The time to cessation of bleeding was monitored. Each data point represents an individual mouse (n=12, 14). B and C , SR-B1/LDLR DKO mice were fed the HFCC for 14 days and treated with the RAG8 or the inactive control SRQ8 pepducins (n=4 each). Platelets were prepared from blood collected retro-orbitally from anesthetized mice. Platelet activation was measured by treatment of platelets with either thrombin or a synthetic PAR4-AP (PAR4 agonist peptide) by flow cytometry after immunostaining with either ( B ) a FITC (fluorescein isothiocyanate)-labeled Wug-E9 antibody (for P-selectin) or ( C ) a PE (phycoerythrin)-labeled JON/A antibody for active GP IIb/IIIa (glycoprotein). Resting platelet activation levels do not differ between control SRQ8- and RAG8-treated SR-B1/LDLR DKO mice. Units are expressed as median fluorescence intensity (MFI). Bars represent means, and error bars represent SEMs. Data in A passed the normality test and were analyzed by Student t test. Data in B and C were analyzed by 2-way ANOVA with Sidak multiple comparisons post hoc test. **** P <0.0001.

Article Snippet: For pepducin-treated mouse groups, platelets (1×10 6 washed mouse platelets per reaction) were resting or activated with 1 U/mL α-thrombin (HT 1002A; Enzyme Research Laboratories, South Bend, IN) or 300 μM PAR4 agonist peptide (AYPGKF-NH 2 ; custom synthesized by Genscript, Piscataway, NJ) in the presence of 2 mM CaCl 2 .

Techniques: Activation Assay, Double Knockout, Control, Saline, Flow Cytometry, Immunostaining, Labeling, Fluorescence