Compositions and methods for inhibiting platelet activation and thrombosis
a technology of platelet activation and inhibition, applied in the field of antithrombotic agents, can solve the problems of thrombotic thrombocytopenic purpura, life-threatening condition, allergic reactions in sensitive individuals, etc., and achieve the effects of improving specific activity, improving specific activity, and binding
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example 1
Effect of Compounds Useful According to the Invention on Agonist-Induced Platelet Activation
[0267] Compounds were tested for the ability to inhibit agonist-induced ADP / ATP release from platelet dense granules using a luciferin-luciferase reporter system. In this assay, compounds were incubated with platelet-rich plasma (PRP) for 30 min. A cocktail of the platelet agonist and luciferin-luciferase was then added to the wells. Plates were immediately analyzed using a Tundra high density luminescence imager. This assay demonstrated a signal to noise ratio of greater than 100:1. As a control, compounds were also screened in a platelet-free assay for the ability to inhibit luciferin-luciferase upon exposure to ATP.
[0268] Each of the compounds was tested for inhibition of platelet activation in response to the agonists SFLLR (a thrombin mimic), U46619 (a thromboxane A2 mimic), ADP and the phorbol ester PMA.
example 2
Effect of Compounds Useful According to the Invention on GPIIbIIIa Activation
[0269] Compounds were also tested by flow cytometry for the inhibition of SFLLR-induced GPIIbIIIa activation and P-selectin expression. In these experiments, freshly obtained PRP was prepared from healthy volunteers who had not ingested aspirin for two weeks prior to donation. Forty μl of PRP was incubated with inhibitor in DMSO or DMSO alone for 15 min. The sample was then incubated with the indicated agonist for 10 min. Following incubation of the sample with the indicated agonist, 10 μl of reaction mixture was transferred to 5 μl of PE-conjugated AC1.2 anti-P-selectin antibody (for assessment of P-selectin surface expression) or FITC-conjugated PAC-1 antibody (for assessment of GPIIbIIIa activation). PBS (500 μl) was added to the sample after a 20 min incubation and the platelets were analyzed immediately by flow cytometry. Flow cytometry was performed using a Becton-Dickinson FACSCalibur flow cytometer...
example 3
Augmentation of PGE1-Induced cAMP Levels in Platelets by Inhibitors Useful According to the Invention
[0270] In order to assess whether the compounds useful according to the invention might act through inhibition phosphodiesterase III, each was tested for its effect on PGE1-induced cAMP increase. Determination of platelet cAMP levels was performed as described by Liao et al., 1998, supra. Aliquots of PRP were exposed to increasing concentrations of the indicated compounds for 20 min. PRP was then challenged with 1 μM PGE1 for 2 min. The reaction was stopped with the addition of 10 mM EDTA followed immediately by boiling for 2 min. The mixture was then cooled to 4° C. and the precipitated protein was pelleted. Cyclic AMP content in the supernatant was then quantified using an enzyme immunoassay kit (Pharmacia-Amersham, N.J.) according to the instructions of the manufacturer. Results are shown in Table III.
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