An optimized protocol to isolate quiescent washed platelets from human whole blood and generate platelet releasate under clinical conditions

The contents of the platelet releasate (PR) play significant roles in hemostasis, inflammation, and pathologic sequelae. Careful platelet isolation to ensure quiescence and subsequent activation is key to the successful generation of PR. Here, we describe steps to isolate and aggregate quiescent washed platelets from whole blood of a clinical patient cohort. We then detail the generation of PR from isolated human washed platelets under clinical conditions. This protocol allows the investigation of platelet cargoes released through various activation pathways. [Display omitted] •Generation of platelet releasate from a clinical patient cohort•Careful isolation of quiescent platelets from whole blood•Demonstration of the effects of medication on platelet aggregation•Applicable for a wide range of patients/diseases Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The contents of the platelet releasate (PR) play significant roles in hemostasis, inflammation, and pathologic sequelae. Careful platelet isolation to ensure quiescence and subsequent activation is key to the successful generation of PR. Here, we describe steps to isolate and aggregate quiescent washed platelets from whole blood of a clinical patient cohort. We then detail the generation of PR from isolated human washed platelets under clinical conditions. This protocol allows the investigation of platelet cargoes released through various activation pathways.