Fig 1.
Losartan dose-dependently and specifically inhibits collagen induced platelet activation.
Washed human platelets were incubated with losartan (A, B,), EXP3179 or EXP3174 (C, D) for 10 min at 37°C. Platelet aggregation (A, C) and P-selectin exposure (A, B, D) were triggered by collagen 1 μg.mL-1 and 10 μg.mL-1, respectively. In (B), washed platelets pre-treated with PBS (open bars) or 22 μM losartan (black bars), were incubated or not with 10 nM angiotensin II for 10 min and then activated by 10 μg.mL-1 collagen before measuring P-selectin exposure. Maximal aggregation is reported (n = 4 for all doses of losartan,); in (A, B and D), P-selectin exposure was measured by flow cytometry and percentage of positive platelets is reported (n = 5 for all doses of losartan). All the data are mean +SEM of n = 4 experiments (platelet aggregation) or n = 5 experiments (P-selectin exposure). Pairwise comparisons were made relative to the condition without losartan; one-way ANOVA followed by a Bonferroni correction was used for multiple comparisons *P<0.05, **P<0.01, ***P<0.001, NS: not significant.
Fig 2.
Losartan reduces thrombus formation on immobilized collagen under flow condition in vitro.
Flow chambers were coated with 50 μg.mL-1 Horm collagen overnight at 4°C. PPACK-anticoagulated whole blood was labeled with 10 μM DiOC6 for 10 min at RT then incubated or not with 22 μM losartan for 10 min at 37°C. Blood was perfused in the flow chambers for 5 min at a shear rate of 1500 s-1 and platelet adhesion was continuously recorded. Images captured at different times after the beginning of the perfusion (A) and of the full length of chambers after 5 min perfusion (B), are from one representative experiment out of three. Quantitative analysis of the mean fluorescence intensity on the whole chamber (C). Data are the mean + SEM of three independent experiments. * P<0.05
Fig 3.
Losartan inhibits collagen induced platelet responses dependent on GPVI and independent on α2β1.
Washed platelets in Mg2+- and Ca2+- free reaction buffer (A) or normal reaction buffer (B-F) were pre-treated with or without 22 μM losartan for 10 min at 37°C. (A) Platelets were incubated in collagen-coated microtiter wells for 40 min and 60 min, respectively. Platelet adhesion was measured by quantifying alkaline phosphatase activity. Data are mean + SEM from 5 independent experiments. (B) Platelets were incubated in GFOGER-coated microtiter wells for 60 min, and adhesion was measured as in (A). Data are mean + SEM from 5 independent experiments. (C) Platelets were incubated in CRP-coated microtiter wells for 40 min and 60 min, and adhesion was measured as in (A). Data are mean + SEM from 6 independent experiments. (D) Platelet aggregation was induced by CRP (0.5 μg.mL-1) and was recorded. Data are expressed as mean + SEM of maximal aggregation from 7 independent experiments. (E and F) P-selectin exposure and αIIbβ3 activation were measured by flow cytometry on CRP-activated platelets. Data are expressed as the median fluorescence intensity (MFI) and are from 6 independent experiments. *P<0.05, **P<0.01, NS: not significant.
Fig 4.
Losartan does not compete with collagen for binding to GPVI.
(A) Washed platelets were treated with PBS or 22 μM losartan or 50 μg.mL-1 Fab 9O12 for 10 min at 37°C. FITC-coupled collagen (10 μg.mL-1) was added and samples were incubated for 20 min at RT and analyzed by flow cytometry. Data are mean + SEM of MFI from 5 independent experiments. (B) The aggregation of control, losartan- or Fab 9O12-treated platelets was triggered by FITC-coupled collagen (1 μg.mL-1). Data are expressed as maximal aggregation. (C) GPVI-Fc (0.25 μg.mL-1) and GPVI-His (10 μg.mL-1) preincubated with increasing concentrations of losartan were added to collagen-coated microtiter wells for 2 h at RT. Bound GPVI was detected using HRP-coupled anti-6×His monoclonal antibody and anti-human IgG, respectively. Data are expressed as mean + SEM from 3 independent experiments.
Fig 5.
Losartan inhibits collagen induced clustering of GPVI but not ADP- or TRAP-induced GPVI dimerization.
Washed platelets preincubated with PBS or losartan (22 μM), were stimulated with collagen (10 μg.mL-1), TRAP (20 μM) or ADP (10 μM) for 20 min. (A) GPVI expression and (B) GPVI dimerization were measured by flow cytometry using FITC-coupled 3J24 and 9E18, respectively. Results (mean + SEM) are from 5 independent experiments. (C) Washed platelets pre-treated with PBS or 22 μM losartan and pre-stimulated or not with TRAP (20 μM) were incubated with FITC-coupled collagen (10 μg.mL-1). Bound collagen was analyzed by flow cytometry. PBS: open bars; losartan: black bars. Data are mean + SEM from 7 independent experiments. * P<0.05, *** P<0.001.
Fig 6.
Losartan inhibits collagen induced platelet activation even when GPVI dimers are preformed.
Washed platelets were preincubated with PBS or losartan (22 μM) for 10 min at 37°C. Buffer or TRAP (10 μM) were added to the samples and the incubation continued without stirring for 10 min. Collagen (1 μg.mL-1) was then added, stirring started and aggregation recorded. (A) Representative aggregation curves. (B) When the aggregation reached 25% in control condition (preincubation with PBS, no losartan), the extent of aggregation in other conditions was measured. Data are represented as bar charts. (Mean + SEM of 3 experiments)
Fig 7.
Duolink imaging of collagen-induced GPVI clustering and its inhibition by losartan.
(A) Washed platelets pretreated with PBS or losartan (22 μM) were incubated with 5 μg.mL-1 9E18-MINUS and 5 μg.mL-1 9E18-PLUS and added to collagen-coated Lab-Tek chambers for 45 min at 37°C. Bound platelets were fixed with 4% PFA and stained with 40 μg.mL-1 anti-CD41 alexa fluo 488 (Green) before ligation and the MINUS and PLUS oligonucleotides and amplification. PLA dots (Orange) are only generated if 9E18-MINUS and—PLUS are in close proximity (<40 nm). Numerous and large GPVI clusters are observed in control conditions while only few spots are visible in the presence of losartan. (B) Quantitative analysis of adherent platelets (green). Data are expressed as the area covered by platelets and the mean + SEM of n = 30 random fields from 3 independent experiments. Scale bars: 5 μm. *P<0.05. (C) Quantitative analysis of platelets GPVI clustering as determined by measuring the proportion of Duolink positive platelets on the same fields.
Table 1.
Platelet functions of patients treated by losartan or placebo.
Fig 8.
Schematic representation of the inhibitory effect of losartan on collagen-induced platelet activation.
Losartan does not impact GPVI dimerization in response to ADP and TRAP but blocks GPVI clustering by collagen and subsequent platelet responses.