A flow cytometric assay for the study of dense granule storage and release in human platelets

Abstract

The clinical manifestations of platelet dense (δ) granule defects are easy bruising, as well as epistaxis and bleeding after delivery, tooth extractions and surgical procedures. The observed symptoms may be explained either by a decreased number of granules or by a defect in the uptake/release of granule contents. We have developed a method to study platelet dense granule storage and release. The uptake of the fluorescent marker, mepacrine, into the platelet dense granule was measured using flow cytometry. The platelet population was identified by the size and binding of a phycoerythrin-conjugated antibody against GPIb. Cells within the discrimination frame were analysed for green (mepacrine) fluorescence. Both resting platelets and platelets previously stimulated with collagen and the thrombin receptor agonist peptide SFLLRN was analysed for mepacrine uptake. By subtracting the value for mepacrine uptake after stimulation from the value for uptake without stimulation for each individual, the platelet dense granule release capacity could be estimated. Whole blood samples from 22 healthy individuals were analysed. Mepacrine incubation without previous stimulation gave mean fluorescence intensity (MFI) values of 83±6 (mean ± 1 SD, range 69–91). The difference in MFI between resting and stimulated platelets was 28±7 (range 17–40). Six members of a family, of whom one had a known δ-storage pool disease, were analysed. The two members (mother and son) who had prolonged bleeding times also had MFI values disparate from the normal population in this analysis. The values of one daughter with mild bleeding problems but a normal bleeding time were in the lower part of the reference interval

Platelets express and release osteocalcin and co‐localize in human calcified atherosclerotic plaques

“…S2B). Moreover, in experiments of labelling with mepacrine, which accumulates preferentially into d-granules of platelets, [18] the signal showed an overall significant decrease after 30 min of stimulation with Ca 2+ -ionophore (Supplementary Matherial, Fig. S2-C).…”

Flow cytometric mepacrine fluorescence can be used for the exclusion of platelet dense granule deficiency

“…Flow cytometry has been recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee guidelines as a tool to diagnose patients with a platelet function disorder, and has been shown to have added value to light transmission aggregometry in diagnosing platelet function disorders. 9,17 Platelet granule markers, such as CD63 and P-selectin, have also been used in the screening of mild platelet function disorders on the flow cytometer, but require platelet stimulation before analysis. Mepacrine, a fluorescent acridine derivative that binds adenosine nucleotides, 18 has been used to measure platelet dense granule content.…”

PIKfyve-Dependent Phosphoinositide Dynamics in Megakaryocyte/Platelet Granule Integrity and Platelet Functions

“…38–40,63,64 Confocal imaging showed that these large vesicles were decorated with the early endosome markers (EEA [early endosome antigen 1] and Rab5) and the MVB/late endosome markers (LAMP 1 and Rab7) while CD63 labeling appeared punctuated at the membrane of the vacuoles and also between vacuoles (Figure 3A). These enlarged vacuoles were also labeled both with AG markers fibrinogen and PF4 and the DG markers mepacrine 65 and LAMP2 (Figure 3B), suggesting a fusion of granules and/or a mis-sorting of cargos during granule biogenesis following PIKfyve inhibition. These enlarged vacuoles co-labeled with AG and DG markers were also observed in MEG-01 treated with another PIKfyve inhibitor, YM201636 (Figure S2A) or displaying a knock-down of PIKfyve (Figures S2B and S5).…”