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

Ramstrom, A. S.; Fagerberga, I. H.; Lindahl, Tomas

doi:10.3109/09537109909169179

Cited by 11 publications

(4 citation statements)

References 2 publications

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“…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).…”

Section: Resultsmentioning

confidence: 99%

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

Strapazzon

Toni

Fabris

et al. 2013

Journal of Thrombosis and Haemostasis

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Summary. Background: Although vascular-calcification mechanisms are only partially understood, the role of circulating calcifying cells and non-collagenous bone matrix proteins in the bone-vascular axis is emerging. In spite of the fact that platelets represent a cellular interface between hemostasis, inflammation and atherosclerosis, and have a myeloid precursor, a possible involvement in the modulation of vascular calcification has rarely been investigated. We investigated if osteocalcin (OC) is released by platelets and described OC expression in patients with carotid artery occlusive disease. Methods: Expression and release of OC were determined by Western blot, immunofluorescence, fluorescence-activated cell sorting (FACS) and ELISA in human resting and activated platelets and megakaryocytes. Co-localization of platelet aggregates, macrophages, OC and calcifications was studied in carotid endarterectomy specimens and normal tissues. Results: Human platelets expressed OC and co-localized with CD63 in d-granules. Upon activation with an endogenous mechanism, platelets released OC in the extracellular medium. Expression of OC in megakaryocytes suggested lineage specificity. The OC count in circulating platelets and the released amount were significantly higher in patients with carotid artery occlusive disease than in healthy controls (P < 0.0001) in spite of similar serum levels. In atherosclerotic plaques, OC strongly overlapped with CD41+ platelets in the early stage of calcification, but this was not seen in normal tissues. CD68+OC+ cells were present at the periphery of the calcified zone. Conclusions: Given the active role played by platelets in the atherosclerotic process, the involvement of OC release from platelets in atherosclerotic lesions and the impact of genetic and cardiovascular risk factors in mediating bone-marrow preconditioning should be investigated further.

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Section: Resultsmentioning

confidence: 99%

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

Strapazzon

Toni

Fabris

et al. 2013

Journal of Thrombosis and Haemostasis

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“…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 . 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.…”

Section: Introductionmentioning

confidence: 99%

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

Asten

Blaauwgeers

Granneman

et al. 2020

Journal of Thrombosis and Haemostasis

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Background δ‐storage pool disease (δ‐SPD) is a bleeding disorder characterized by a reduced number of platelet‐dense granules. The diagnosis of δ‐SPD depends on the measurement of platelet ADP content, but this test is time consuming and requires a relatively large blood volume. Flow cytometric analysis of platelet mepacrine uptake is a potential alternative, but this approach lacks validation, which precludes its use in a diagnostic setting. Objectives To evaluate the performance of platelet mepacrine uptake as a diagnostic test for δ‐SPD. Patients/Methods Mepacrine fluorescence was determined with flow cytometry before and after platelet activation in 156 patients with a suspected platelet function disorder and compared with platelet ADP content as a reference test. Performance was analyzed with a receiver operating characteristic (ROC) curve. Results Eleven of 156 patients had δ‐SPD based on platelet ADP content. Mepacrine fluorescence was inferior to platelet ADP content in identifying patients with δ‐SPD, but both mepacrine uptake (area under the ROC curve [AUC] 0.87) and mepacrine release after platelet activation (AUC 0.80) had good discriminative ability. In our tertiary reference center, mepacrine uptake showed high negative predicitive value (97%) with low positive predictive value (35%). Combined with a negative likelihood ratio of 0.1, these data indicate that mepacrine uptake can be used to exclude δ‐SPD in patients with a bleeding tendency. Conclusion Mepacrine fluorescence can be used as a screening tool to exclude δ‐SPD in a large number of patients with a suspected platelet function disorder.

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“…Hence, using flow cytometry, we gated CD41+ cells (platelet positive staining), and fluorescence of anti‐P‐selectin (CD62P) antibody and quinacrine was monitored. Fluorescence protocols have been widely used to evaluate alpha (α‐) and dense (δ‐) granule secretion . As shown in Figure A, platelets present low levels of surface‐exposed P‐selectin under resting conditions (Fig.…”

Section: Resultsmentioning

confidence: 99%

Long‐term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients

López

Berna‐Erro

Bermejo

et al. 2013

J Cellular Molecular Medi

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The use of the mammal target of rapamycin (mTOR) inhibitors has been consolidated as the therapy of election for preventing graft rejection in kidney transplant patients, despite their immunosuppressive activity is less strong than anti-calcineurin agents like tacrolimus and cyclosporine A. Furthermore, as mTOR is widely expressed, rapamycin (a macrolide antibiotic produced by Streptomyces hygroscopicus) is recommended in patients presenting neoplasia due to its antiproliferative actions. Hence, we have investigated whether rapamycin presents side effects in the physiology of other cell types different from leucocytes, such as platelets. Blood samples were drawn from healthy volunteers and kidney transplant patients long-term medicated with rapamycin: sirolimus and everolimus. Platelets were either loaded with fura-2 or directly stimulated, and immunoassayed or fixed with Laemmli's buffer to perform the subsequent analysis of platelet physiology. Our results indicate that rapamycin evokes a biphasic time-dependent alteration in calcium homeostasis and function in platelets from kidney transplant patients under rapamycin regime, as demonstrated by the reduction in granule secretion observed and subsequent impairment of platelet aggregation in these patients compared with healthy volunteers. Platelet count was also reduced in these patients, thus 41% of patients presented thrombocytopenia. All together our results show that long-term administration of rapamycin to kidney transplant patients evokes alteration in platelet function.

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

Cited by 11 publications

References 2 publications

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

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

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

Long‐term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients

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