Figure 1.
In silico identifies GPVI antagonists.
Representative image capture of in silico docking into GPVI using Glide, with space filling model is shown in a, and H-bonding to relevant side chains is detailed in b. The 20 highest ranking compounds were screened for effects on Ca2+ release by the GPVI-specific agonist CRP-XL (10 µg/ml) (c and d, % refers to percent inhibition of Ca2+ release). Maximum Ca2+ release is shown in white, compounds that inhibited Ca2+ release by ∼50% or more are in grey, and the remainder in black. Commercially available compounds that inhibited CRP-XL-induced Ca2+ release >50% were further screened by light transmission aggregometry to identify compounds displaying dose-dependent inhibition (e-j). Examples are shown of weak antagonism (g and h) and false positives (i and j). Cinanserin (l) and losartan (k) were taken on for further study.
Figure 2.
Losartan and cinanserin inhibit GPVI-mediated cell activation.
Washed human platelets were loaded with fura2-AM and screened for drug-mediated inhibition of Ca2+ release by 1 µg/ml CRP-XL (n = 3, ± SEM, representative traces and summary, a–c) and 1 µg/ml collagen (n = 3, SEM, representative traces and summary, d–f); losartan (▪) and cinanserin (▴). To measure aggregation, washed human platelets were incubated with drug for one minute prior to the addition of 1 μg/ml CRP-XL (representative traces and summary shown in g–i) or 1 µg/ml collagen (representative traces and summary shown in j–l).
Table 1.
IC50 values for losartan and cinanserin on both CRP-XL- and collagen-induced Ca2+ release and aggregation.
Figure 3.
Losartan and cinanserin demonstrate selectivity for GPVI.
Ca2+ release and aggregations were carried out with 5 μM of the P2Y12 receptor agonist 2-MeSADP (a, Ca2+ release and b, aggregation), or 0.5 U/ml of the PAR1 and PAR4 receptor agonist thrombin (c, Ca2+ release and d, aggregation). Losartan (⧫); cinanserin (▴), n = 3, ± SEM. For global tyrosine phosphorylation, washed human platelets were incubated with drug or vehicle alone before addition of 1 µg/ml CRP-XL or collagen. Samples were collected at 10, 30, 60 or 90 seconds (as indicated by the graduated bars with time increasing to the right) in ice cold 2× lysis buffer and separated on 4–12% NuPage gels under reducing conditions. Tyrosine phosphorylation was visualized with 4G10 anti-phosphotyrosine antibody. Losartan and cinanserin reduce CRP-XL- (e) and collagen-(f) induced global tyrosine phosphorylation, but have no effect on thrombin or 2-MeSADP induced global tyrosine phosphorylation (g). Both drugs reduce FcRγ phosphorylation (h), (unphosphorylated, lower band; phosphorylated, upper band).
Figure 4.
Losartan is selective for GPVI over TPR.
To assess effects on TPR signalling, platelets were activated with 1 µM U46619 and aggregations followed for 5 minutes in the presence of or absence of drug (n = 3, ± SEM). Collagen (solid black line) and CRP-XL (dashed dark grey line) were both used at 1 µg/ml. Losartan (a) has an IC50 of ∼20 µM for U46619-induced aggregation (solid grey line) while cinanserin (b) has no effect on TPR signaling (solid grey bar). Exposure of P-selectin (CD62P) and FITC-fibrinogen binding was measured by flow cytometry. Losartan (100 µM, c) and cinanserin (100 µM, d) both reduce FITC-fibrinogen binding (black bars) and P-selectin exposure (grey bars) compared to SQ-29548 alone. Statistical analysis was conducted by one-way ANOVA with Sidak's multiple comparisons correction. ns P>0.05, * P<0.05, *** P<0.001, n = 3, ± SEM.
Figure 5.
Losartan displays characteristics of competitive antagonists.
Platelet aggregation responses to the GPVI-specific agonist CRP-XL were determined to a range of concentrations in the presence or absence of 30 µM drug. Losartan (a) significantly reduced the EC50 of CRP (F(1,58) = 15.79, p = <0.001). Cinanserin (b) also reduced the EC50 of CRP (F(1,60) = 4.07, p = 0.048).
Figure 6.
GPVI antagonism is unique to losartan amongst the sartan drug class.
Inhibition of collagen-induced Ca2+ release (10 µg/ml) the sartans from fura2-AM-loaded washed human platelets was measured and quantified (n = 3, ± SEM).