Platelet isolation and activation assays

Abstract

Platelets regulate hemostasis and are the key determinants of pathogenic thrombosis following atherosclerotic plaque rupture. Platelets circulate in an inactive state, but become activated in response to damage to the endothelium, which exposes thrombogenic material such as collagen to the blood flow. Activation results in a number of responses, including secretion of soluble bioactive molecules via the release of alpha and dense granules, activation of membrane adhesion receptors, release of microparticles, and externalization of phosphatidylserine. These processes facilitate firm adhesion to sites of injury and the recruitment and activation of other platelets and leukocytes, resulting in aggregation and thrombus formation. Platelet activation drives the hemostatic response, and also contributes to pathogenic thrombus formation. Thus, quantification of platelet-Associated responses is key to many pathophysiologically relevant processes. Here we describe protocols for isolating, counting, and activating platelets, and for the rapid quantification of cell surface proteins using flow cytometry.