ml161 par-1 inhibitor  (Cayman Chemical)

Journal: Thrombosis and haemostasis

Article Title: P2Y12 Inhibition Suppresses Proinflammatory Platelet-Monocyte Interactions

doi: 10.1055/s-0042-1758655

Figure Lengend Snippet: (A) Platelet releasates (PR) were generated from whole blood samples incubated for 30 min with platelet inhibitors (50 μM ML161, 1 μM AZD1283, 18 μM eptifibatide, or 1 mM aspirin) before platelet-rich plasma was generated by centrifugation. Platelets were then transferred to Tyrode’s buffer supplemented with Ca2+ and platelet releasates were collected following high-speed centrifugation of platelet resuspension. For monocyte-platelet releasate incubation assays, CD14+ selected human monocytes were incubated with platelet releasates for 6 h. Monocytes were then lysed and RNA was collected for RT-qPCR analyses. WB = whole blood, PRP = platelet-rich plasma. (B) Monocyte mRNA expression of SOCS3 and (D) OSM compared to nontreated monocytes. Expression of SOCS3 (C) and OSM (E) following exposure of CD14 isolated human monocytes to platelet releasates generated in the presence of PAR1 inhibitor ML161 (50 μM), P2Y12 inhibitor AZD1283 (1 μM), COX-1 inhibitor aspirin (1 mM), and GP IIb/IIIa inhibitor eptifibatide (18 μM) for 6h (right panels). Data expressed relative to platelet releasate-treated cells in the absence of platelet inhibitors (n = 7 unique releasate donors; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by a paired t-test).

Article Snippet: To assess platelet inhibitors, PRP was incubated with ML161 (PAR-1 inhibitor; 10 μM, 50 μM, Cayman Chemicals), AZD1283 (P2Y 12 inhibitor; 0.1 μM, 1 μM, 10 μM, Cayman Chemicals), eptifibatide (glycoprotein [GP] IIb/IIIa inhibitor; 18 μM, Cayman Chemicals), or aspirin (1 mM, Santa Cruz Biotechnology) for 30 min at 37°C before being activated with either thrombin (0.5 U/mL, Werfen), ADP (1 μM, Helena), or arachidonic acid (160 μM, Helena) for 90 seconds ( S Table 2 ).

Techniques: Generated, Incubation, Clinical Proteomics, Centrifugation, Quantitative RT-PCR, Expressing, Isolation

Journal: Thrombosis and haemostasis

Article Title: P2Y12 Inhibition Suppresses Proinflammatory Platelet-Monocyte Interactions

doi: 10.1055/s-0042-1758655

Figure Lengend Snippet: All experiments were performed in citrate-anticoagulated whole blood. Monocyte-platelet aggregates (MPA, %) were assessed by quantification of CD61+ event of CD45+CD14+CD16+ monocytes by flow cytometry. (A) MPA formation following stimulation for 30 min with U-46619 in a final concentration of 0.56 μM (red bar) compared to a nonstimulated control. P-value determined by unpaired t-test. (B) Whole blood was incubated with platelet inhibitors anti-P-selectin (0.5 μg/ml), anti-PSGL1 (0.5 μg/ml), or (C) PAR1 inhibitor (ML161, 50 μM), P2Y12 inhibitor (AZD 1283, 1 μM), GP IIb/IIIa inhibitor (eptifibatide, 18 μM), or aspirin (COX-1, 1 mM) for 30 min, followed by U-46619 stimulation (0.56 μM) for 30 min. MPA formation of U-46619-stimulated whole blood that had been pretreated with different platelet inhibitors was compared to MPA formation of stimulated controls set as 100% (as shown as a dashed line). P-values determined by one sample t-tests. All data presented as *p<0.05, **p<0.01, ***p<0.001, ****p< 0.0001.

Article Snippet: To assess platelet inhibitors, PRP was incubated with ML161 (PAR-1 inhibitor; 10 μM, 50 μM, Cayman Chemicals), AZD1283 (P2Y 12 inhibitor; 0.1 μM, 1 μM, 10 μM, Cayman Chemicals), eptifibatide (glycoprotein [GP] IIb/IIIa inhibitor; 18 μM, Cayman Chemicals), or aspirin (1 mM, Santa Cruz Biotechnology) for 30 min at 37°C before being activated with either thrombin (0.5 U/mL, Werfen), ADP (1 μM, Helena), or arachidonic acid (160 μM, Helena) for 90 seconds ( S Table 2 ).

Techniques: Flow Cytometry, Concentration Assay, Control, Incubation