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, 148 (1), 61-9

Biological Properties of a Specific Galpha q/11 Inhibitor, YM-254890, on Platelet Functions and Thrombus Formation Under High-Shear Stress

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Biological Properties of a Specific Galpha q/11 Inhibitor, YM-254890, on Platelet Functions and Thrombus Formation Under High-Shear Stress

Toshio Uemura et al. Br J Pharmacol.

Abstract

1 The effects of YM-254890, a specific Galpha(q/11) inhibitor, on platelet functions, thrombus formation under high-shear rate condition and femoral artery thrombosis in cynomolgus monkeys were investigated. 2 YM-254890 concentration dependently inhibited ADP-induced intracellular Ca(2+) elevation, with an IC(50) value of 0.92+/-0.28 microM. 3 P-selectin expression induced by ADP or thrombin receptor agonist peptide (TRAP) was strongly inhibited by YM-254890, with IC(50) values of 0.51+/-0.02 and 0.16+/-0.08 microM, respectively. 4 YM-254890 had no effect on the binding of fibrinogen to purified GPIIb/IIIa, but strongly inhibited binding to TRAP-stimulated washed platelets. 5 YM-254890 completely inhibited platelet shape change induced by ADP, but not that induced by collagen, TRAP, arachidonic acid, U46619 or A23187. 6 YM-254890 attenuated ADP-, collagen-, TRAP-, arachidonic acid- and U46619-induced platelet aggregation with IC(50) values of <1 microM, whereas it had no effect on phorbol 12-myristate 13-acetate-, ristocetin-, thapsigargin- or A23187-induced platelet aggregation. 7 High-shear stress-induced platelet aggregation and platelet-rich thrombus formation on a collagen surface under high-shear flow conditions were concentration dependently inhibited by YM-254890. 8 The antithrombotic effect of YM-254890 was evaluated in a model of cyclic flow reductions in the femoral artery of cynomolgus monkeys. The intravenous bolus injection of YM-254890 dose dependently inhibited recurrent thrombosis without affecting systemic blood pressure or prolonging template bleeding time. 9 YM-254890 is a useful tool for investigating Galpha(q/11)-coupled receptor signaling and the physiological roles of Galpha(q/11).

Figures

Figure 1
Figure 1
Chemical structure of YM-254890.
Figure 2
Figure 2
Effects of YM-254890 (a) and MRS2179 (b) on ADP-induced human platelet intracellular Ca2+ elevation. Fura-2-loaded platelets were stimulated with ADP (10 μM) 1 min after the addition of test compounds. The data are representative of three separate experiments.
Figure 3
Figure 3
Effects of YM-254890 and MRS2179 on P-selectin expression and fibrinogen binding. P-selectin expression was evaluated in 5 μM ADP- (a) or 20 μM TRAP- (b) stimulated human platelets using flow cytometry. Fibrinogen binding (c) was measured using human PRP stimulated with 20 μM TRAP. Data represent the mean±s.e.m. (n=3–5). Statistical analyses were performed using the Student's t-test for comparison with unstimulated platelets or Dunnett's multiple comparison test for comparison with agonist-stimulated platelets. ##P<0.01 compared with the unstimulated platelets. *P<0.05 and **P<0.01 compared with the agonist-stimulated platelets.
Figure 4
Figure 4
Effect of YM-254890 on human platelet shape change after the addition of various agonists. During the analysis of platelet shape change, platelet aggregation was blocked by the addition of abciximab (50 μg ml−1). At 1 min after the addition of the test sample to PRP in an aggregometer at 37°C, followed ADP (20 μM), collagen (1 μg ml−1), TRAP (5 μM), arachidonic acid (1.5 mM), U46619 (3 μM) or A23187 (5 μM), as shown with arrows. YM-254890 was used at a final concentration of 10 μM for all agonists. The data are representative of three separate experiments.
Figure 5
Figure 5
Effect of YM-254890 on SIPA in human PRP. PRP was exposed to a gradient of 6 × 10−5 to 1.08 × 10−3N cm−2 for 6 min at room temperature. After an initial 15 s at 6 × 10−5N cm−2, the gradient increased from 6 × 10−5 to 1.2 × 10−4N cm−2 over 90 s, from 1.2 × 10−4 to 1.08 × 10−3N cm−2 over the next 120 s, and remained constant at 1.08 × 10−3N cm−2 for the last 135 s. The data are representative of three separate experiments. The YM-254890 concentrations used are indicated.
Figure 6
Figure 6
Effect of YM-254890 on human platelet thrombus formation on a collagen surface in a perfusion chamber. (a) Three-dimensional images of platelet thrombus formation on a collagen surface in the presence of YM-254890 under high-shear rate conditions (1500 s−1). The three-dimensional images of thrombus generation on a collagen surface 7 min after the beginning of the perfusion were digitized, and the three-dimensional structures were constructed based on the two-dimensional images using commercial volume analysis software. Top and bottom panels show platelet thrombus images from the horizontal and oblique positions, respectively. (b) The inhibitory effect of YM-254890 on thrombus formation on a collagen surface under high-shear rate conditions. Data are expressed as percent of the thrombus volume formed in the presence of vehicle, and the data represent the mean±s.e.m. of five separate experiments. Statistical analyses were performed using Dunnett's multiple comparison test for comparison with the vehicle-treated blood samples. **P<0.01 compared with the vehicle group.
Figure 7
Figure 7
The femoral artery thrombosis model in cynomolgus monkeys. (a) Typical patterns of CFRs, HR and BP after i.v. bolus injection of saline and YM-254890. CFRs were induced in the femoral artery by stenosis and injury. (b) Effect of YM-254890 on platelet thrombus formation. Open circles represent the CFR rating on a scale of 0–3, with 0 indicating no inhibition and 3 indicating complete inhibition. Bars indicate the median values for each dose. (c) Effect of YM-254890 on ex vivo ADP-induced platelet aggregation. Data are expressed as the mean±s.e.m. of five animals. Statistical analysis was performed using Dunnett's multiple comparison test. **P<0.01 compared with the saline group. (d) Effect of YM-254890 on bleeding time. Open circles represent the bleeding time for each animal. Bars indicate the median values for each dose. Statistical analysis was performed using Steel's test. (e) Effect of YM-254890 on relative change in HR (closed circles) and BP (closed triangles). Data are expressed as the mean±s.e.m. of five animals. Statistical analysis was performed using Dunnett's multiple comparison test.

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