Key Points• Low-volume, high-throughput whole blood aggregometry will facilitate future mouse platelet function research.• Application of this approach identifies ICAM-1 as a novel mediator of platelet-monocyte interaction through fibrinogen binding.Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation. (Blood. 2015;126(10):e11-e18)
IntroductionPlatelets are key mediators of hemostasis. They have crucial roles in certain bleeding disorders and are the targets of antithrombotic therapies, notably aspirin and P2Y 12 receptor blockers such as clopidogrel. [1][2][3] Numerous assays have been developed to investigate platelet responses with an understandable focus on analyzing human clinical samples. 4,5 These assays require relatively large volumes of blood, with the majority requiring a sample volume in excess of 200 mL per test. 6 Mouse models are widely used in cardiovascular research, particularly for phenotyping of genetic modifications and testing novel therapeutic agents. However, platelet function testing in mice remains difficult because a complete and terminal blood collection provides at most 1 mL of blood. Further, commonly used strategies for extending the volume available such as dilution or platelet washing can introduce artifactual changes in platelet responses and thus does not permit study of platelet interactions with other blood cells and constituents. As a result, both the number and type of platelet tests that can be performed in samples from mice are severely limited, diminishing the experimental value of each mouse and making detailed study of mouse platelets impractical. There is therefore a clear and pressing experimental demand for murine-focused and optimized ...