Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies.

Abstract

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated in metabolic disorders, increasing risk for both thrombosis and bleeding. Platelet activation and metabolism are tightly linked, with the numerous methods to measure the former but relatively few for the latter. To study platelet metabolism without the interference of other blood cells and plasma components, platelets must be isolated, a process that is not trivial because of platelets shear sensitivity and ability to irreversibly activate. Presented here is a protocol for platelet isolation (washing) that produces quiescent platelets that are sensitive to stimulation by platelet agonists. Successive centrifugation steps are used with the addition of platelet inhibitors to isolate platelets from whole blood and resuspend them in a controlled, isosmotic buffer. This method reproducibly produces 30%-40% recovery of platelets from whole blood with low activation as measured by markers of granule secretion and integrin activity. Platelet count and fuel concentration can be precisely controlled to allow the user to probe a variety of metabolic situations.