Fig 1.
PL inhibited collagen-induced platelet reactivity.
(A) Platelets were induced to aggregate by 5 μg/ml of collagen in the presence of increasing concentrations of PL (a representative of 10 separate experiments). (B) PL blocked platelet aggregation induced by low doses, but not a maximal dose of collagen (n = 10, paired t test). (C) PL blocked the thrombus formation of platelets on the collagen matrix under flow conditions (panels a-e are representative images [bar = 100 μm] and panel f is a summary of 6 separate experiments, repeated measures ANOVA, * p < 0.01 compared untreated platelets). PL blocked collagen-induced CD62p expression (D), PAC-1 binding (E), and calcium influx (F). Data presented in panels D-F were obtained from 6–8 donors and analyzed by ANOVA (panels D-E) and t test (panel F).
Fig 2.
Effects of PL on collagen-induced platelet activation, morphological transition, and microvesiculation.
PRP was stimulated with 5 μg/ml of collagen in the presence and absence of 100 μM PL. Surface expressions of GP VI (A, n = 5, ANOVA) and αIIbßß3 (B, n = 6, * p < 0.001 compared to PL treated platelets) were measured by flow cytometry using specific monoclonal antibodies. (C) PL dose-dependently blocked CRP-induced platelet aggregation (n = 6, ANOVA). The morphological transition of adherent platelets on the collagen matrix was monitored by HPICM over ~60 min (D & E, bar = 10 μm). A ratio of HDBS to LDSS platelets was calculated (F, n = 6, paired t test) and CD42b+ and annexin V+ platelet microparticles were quantified (G, n = 6, paired t test).
Fig 3.
PL blocked JAK2-STAT3 activation.
Lysates of platelets stimulated with collagen in the presence of PL were probed for total and phospho-JAK2 (A) and STAT3 (B). STAT3 phosphorylation was also probed in platelets treated with a complex of IL-6-sIL-6α either alone or combined with collagen as control (B). Platelet aggregation was also induced by collagen in the presence of a submaximal 50 μM of PL with and without a maximal inhibitory 50 μM of STA21 (C, a representative of 6 experiments) or actinomycin D (D, a representative of 6 experiments). Platelets treated with an increasing concentration of the JAK2 inhibitor AG490 were induced to aggregate (E) and to form thrombi on immobilized collagen under flow condition (F, bar = 100 μm, the panel e summaries results from 3 experiments, repeated measure ANOVA, *p < 0.01).
Fig 4.
PL effect on collagen-GP VI signaling.
Lysates of platelets treated with collagen in the presence of PL were probed for total and phosphorylated Syk (A) and PLCγ2 (B). (C) Platelets were first incubated with SykII for 10 min followed by additional 10 min incubation with PL before being stimulated with collagen (n = 3, repeated measures ANOVA, *p < 0.01).
Fig 5.
PL-induced ROS production and activity.
Platelets in PRP were labeled with either CellROX Red or DCFH-DA for 30 min at 37°C and treated with either100 μM of PL or vehicle control (BL) for additional 10 min. ROS production was measured in platelets by flow cytometry (A, n = 10, paired t test). ROS production was also measured in platelets stimulated with various doses of collagen in the presence and absence of PL (B, n = 6, paired t test). Free GSH was measured by mass spectrometry in resting platelets and those treated with either PL or collagen for 10 min at 37°C (C, n = 3, one way ANOVA). Collagen-induced aggregation was recorded in platelets preincubated with either GSH (D) or L-cysteine before treatment with 100 μM PL (E). Platelets treated with GSH or L-cysteine without PL were tested as controls (panel F summaries data from 3 experiments). Platelets were treated with collagen, PL, and PL plus GSH for 10 min at 37°C. PAC-1 binding was then detected by flow cytometry (G, a representative of 3 experiments). PRP was incubated with Apocynin for 10 min at 37°C followed by additional 10 min of incubation with 100 μM of PL. Collagen-induced platelet aggregation was then recorded (H, a representative of 3 experiments).
Fig 6.
Effects of PL analogs on collagen-induced platelet aggregation.
(A) PL and structural derivatives compound 1 (C1) and compound 2 (C2). (B) PRP was incubated with PL, C1 or C2 for 15 min at 37°C and then induced to aggregate by 2 μg/ml of collagen (a representative of 10 experiments). (C & D) C1 and C2 were also tested at different doses for their impact on collagen-induced platelet aggregation.