Can the antiplatelet effects of cangrelor be reliably studied in mice under in vivo and in vitro conditions using flow cytometry?

Background: The effects of blood platelet inhibitors are often not quite equivalent under in vivo and in vitro conditions. Amongst various models of human pathology using laboratory animals, mice offer several benefits that make them convenient tools for studying the putative therapeutic value of various compounds. However, despite its advantages, the mouse model has methodological limitations concerning the small amount of blood available and technical difficulties with its collection. Among the variety of available methods used to study blood platelet activation and/or reactivity, flow cytometry seems an attractive technique that largely minimizes the constraints of using small rodents and enables outcomes of laboratory research to be transferred successfully to clinical practice. In this study we aimed at a critical evaluation of the optimal discriminative flow cytometric protocol, useful for reliable studies of the effect of cangrelor, a P2Y(12) receptor antagonist, on mouse platelets under in vitro and in vivo conditions. Methods: Blood samples were drawn from two-month-old female BALB/c mice. Protocols differing in methods of anesthesia, blood withdrawal, anticoagulation, gating antibodies, blood preparation and fixation were tested to optimize the one best suited to discrimination between resting and activated platelets. The antiplatelet capabilities of cangrelor were tested in vitro (140 mu M in whole blood) and in vivo (7.8 mg/kg b.w. administered once, directly into the bloodstream through the vena cava of the anesthetized animal, 15 min prior to blood withdrawal). Expressions of P-selectin, activated alpha(IIb)beta(3) complex and GPIb alpha were monitored using two-color flow cytometry. Results: "Washed blood" anticoagulated with low molecular weight heparin demonstrated the best discrimination between circulating (resting) platelets and upon their in vitro response to thrombin, collagen or ADP in freshly-stained unfixed cell suspensions. Cangrelor inhibited the expression of the active form of the integrin alpha(IIb)beta(3) to approximately the same extent under in vitro and in vivo conditions (84.5 +/- 7.7% vs. 75.4 +/- 19.5% for the in vitro and in vivo approaches, respectively, n.s.). Conclusions: The agreement between the in vivo and in vitro approaches with respect to cangrelor-inhibited hallmarks of blood platelet activation and reactivity supports our proposal that flow cytometry is useful and reliable for determining the effects of antiplatelet agents on the activation of circulating platelets in the mouse model, as well as the in vitro response of platelets to agonists.