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.

Extensive aggregation of human platelets can be induced by ADP without secondaryaggregation or release of granule contents. This occurs with washed platelets in Tyrode solution containing 0.35% albumin, human fibrinogen, and apyrase, and in platelet-rich, heparin- or hirudin-plasma. Conditions that caused release during ADP-inducedaggregation were-citrate as the anticoagulant in platelet-rich plasma; addition of citrate (11-15 mM) to a suspension of washed platelets, or to hirudin-plasma or heparin-plasma; suspension of platelets in a medium containing magnesium but no calcium;and the presence of trace amounts of thrombin or aggregated gamma globulin in the platelet suspensions. Acetylsalicylic acid, phenylbutazone, or sulfinpyrazone inhibited secondary aggregation and release in all these circumstances. Heparin or hirudin inhibited ADP-INDUCED SECONDARY AGGREGATION AND RELEASE PROMOTED BY TRACES OF THROMBIN. Although fibrinogen is required for ADP-induced primary aggregation, it does not support secondary aggregation and release, provided that it has no clot-promoting activity. The main agent responsible for ADP-induced secondary aggregation and release in human, citrated, platelet-rich plasma appears to be sodium citrate. Suspending washed human platelets in a medium without calcium mimics the effect of citrate.

A method is described for the preparation of suspensions of washed rabbit platelets which will aggregate upon the addition of low concentrations of ADP that are effective in citrated plasma. This method involves collecting blood into ACD, low pH, removal of calcium, and maintenance of magnesium during the isolation procedure. A protein (albumin) and glucose are included in the washing and suspending solutions. Rabbit platelets prepared in this way retained fibrinogen on their surface. On comparison with rabbit platelets prepared from blood taken into EDTA, the only differences observed were the much greater sensitivity to ADP and a higher calcium content ; platelet morphology, nucleotide levels, and conversions of 14C-ATP and 14C-ADP at the platelet membrane were similar.