Conditions influencing release of granule contents from human platelets in citrated plasma induced by ADP or the thrombin receptor activating peptide SFLLRN: Direct measurement of percent release of β‐thromboglobulin and assessment by flow cytometry of P‐selectin expression

Abstract

Contrary to a recent report [Rinder et al.: Blood 82:505, 1993], aspirin does inhibit the release of α-granule contents as well as inhibiting the release of dense granule contents by human platelets during ADP-induced aggregation in citrated platelet-rich plasma (PRP). Measurements were: percent release of 14C-serotonin from prelabeled platelets, radio-immunoassay of β-thromboglobulin (βTG), and expression on the platelet surface of the α-granule constituent, P-selectin, by flow cytometry. During the second phase of ADP-induced aggregation, 69.0 ± 8.3% of βTG and 54.1 ± 4.6% of 14C-serotonin were released (means ± SEM, n = 13); aspirin treatment reduced these values to 6.0 ± 1.2 and 1.0 ± 0.3%, respectively. In contrast, incubation of platelets with ADP without stirring caused only 6.7 ± 1.7% release of βTG and 2.1 ± 0.4% release of 14C-serotonin; these low values were not appreciably affected by aspirin. During ADP-induced primary aggregation in PRP anticoagulated with FPRCH2Cl (PPACK), only 4.7 ± 0.9% release of βTG and no detectable release of 14C-serotonin occurred; aspirin had no effect. In both stirred and unstirred PRP, the thrombin receptor activating peptide, SFLLRN (50 μM), caused at least 75% release of the contents of both granules, which was partially inhibited by aspirin. Upon incubation of platelets with ADP (2–10 μM), the mean fluorescence intensity due to P-selectin was <14% of that induced by SFLLRN. In this unstirred system used for flow cytometry, aspirin treatment caused no significant inhibition of P-selectin expression. Thus, under conditions in which ADP does not cause secondary aggregation (physiological Ca2??? concentration or unstirred citrated PRP) release of the contents of both types of granules is less than 7% and aspirin is not inhibitory; the P-selectin expression associated with this low percent release is also unaffected by aspirin. However, aspirin does strongly inhibit the extensive release of both α-granule and dense granule contents during ADP-induced secondary aggregation in citrated PRP. © 1996 Wiley-Liss, Inc.