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 beta-thromboglobulin and assessment by flow cytometry of P-selectin expression.
Contrary to a recent report [Rinder et al.: Blood 82:505, 1993], aspirin does inhibit the release of alpha-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 beta-thromboglobulin (beta TG), and expression on the platelet surface of the alpha-granule constituent, P-selectin, by flow cytometry. During the second phase of ADP-induced aggregation, 69.0 +/- 8.3% of beta TG and 54.1 +/- 4.6% of 14C-serotonin were released (mean +/- 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 beta TG 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 FPRCH2CI (PPACK), only 4.7 +/- 0.9% release of beta TG 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 microM), 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 microM), 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 alpha-granule and dense granule contents during ADP-induced secondary aggregation in citrated PRP.
Rand
M L
ML
Department of Biochemistry, University of Toronto, Ontario, Canada.
Perry
D W
DW
Packham
M A
MA
Gemmell
C H
CH
Yeo
E L
EL
Kinlough-Rathbone
R L
RL
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D001241:Aspirin / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002951:Citrates; D019343:Citric Acid; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D019007:P-Selectin / Q000032:analysis*; D010446:Peptide Fragments / Q000494:pharmacology*; D010949:Plasma; D018179:Receptors, Thrombin / Q000187:drug effects* / Q000502:physiology; D012701:Serotonin / Q000097:blood; D001620:beta-Thromboglobulin / Q000378:metabolism*
D002951:Citrates; D019007:P-Selectin; D010446:Peptide Fragments; D018179:Receptors, Thrombin; D001620:beta-Thromboglobulin; C082835:thrombin receptor peptide (42-47); D019343:Citric Acid; D012701:Serotonin; D000244:Adenosine Diphosphate; D001241:Aspirin