Preparation of Suspensions of Washed Platelets from Humans

TLDR

Methods were developed to prepare suspensions of washed platelets from human donors. The protocol employs heparin to inhibit thrombin generation, apyrase to prevent adenine nucleotide accumulation, and fibrinogen to maximize ADP‑induced aggregation during platelet washing. Platelets suspended in Eagle’s medium with albumin respond more strongly to ADP than those in Tyrode’s‑albumin solution, remain aggregable for at least four hours at 37 °C when apyrase is present, and lose ADP sensitivity without apyrase, which can be partially restored by adding apyrase.

Abstract

Summary: Methods have been developed for the preparation of suspensions of washed platelets from humans. Heparin is used in the washing fluids to prevent: thrombin generation and apyrase is used to prevent adenine nucleotide accumulation. Platelets suspended in Eagle's tissue culture medium containing albumin were more responsive to ADP than platelets in Tyrode's‐albumin solution. Addition of fibrinogen is required for maximum sensitivity to ADP‐induced aggregation. These platelets can be stored for 4 hr or more at 37°C in the presence of apyrase and maintain their ability to aggregate upon the addition of low concentrations of ADP. Without apyrase the platelets gradually become insensitive to ADP upon storage at 37°C; this is presumably caused by the accumulation of ADP in the suspending fluid because sensitivity can be partially restored by the addition of apyrase and further incubation.