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4 results on '"Stefanie Uhlig"'

1. Expression of ADP receptor P2Y12, thromboxane A2 receptor and C-type lectin-like receptor 2 in cord blood-derived megakaryopoiesis

Author

Catharina Gerhards, Stefanie Uhlig, Mani Etemad, Foteini Christodoulou, Karen Bieback, Harald Klüter, and Peter Bugert

Subjects
inherited platelet disorder, megakaryopoiesis, platelet function, receptor expression, Diseases of the blood and blood-forming organs, RC633-647.5
Abstract

The ADP receptor P2Y12, the thromboxane A2 receptor (TXA2R) and the C-type lectin-like receptor 2 (CLEC-2) mediate platelet activation by different mechanisms. Only little is known about the expression of the receptors in human megakaryopoiesis. Our study aimed to establish a flow cytometry (FC) method for the measurement of P2Y12, TXA2R, and CLEC-2 on platelets of healthy donors and to monitor receptor expression in ex vivo megakaryopoiesis. We determined mean fluorescence intensity (MFI) values of FITC, PE, or APC labeled antibodies binding to the receptors on platelets of 90 healthy donors. For cord blood-derived megakaryopoiesis (CBMK) differentiation of CD34+ cells was induced by IL-3, SCF, and TPO. At 6 time points between day 0 and day 21 of cell culture the MFI values for CD34, CD41, CD61, P2Y12, TXA2R, and CLEC-2 were measured. Quantitative PCR was used for relative quantification of the corresponding mRNA. Transcription factor (TF) binding sites were predicted by in silico analysis of the genes. Platelets showed expectable high MFI values for the platelet marker CD41 (13,716 median MFI). Lower MFI was found for P2Y12 (2,847 median MFI) and CLEC-2 (1,211 median MFI), whereas, binding of the TXA2R antibody revealed even higher values (21,458 median MFI) than CD41. In CBMK the CD34+ cells were negative for P2Y12, TXA2R, and CLEC-2 at day 0. A maximum of 21-fold and 6-fold increase of P2Y12 and TXA2R MFI values, respectively, was found on day 14 to 17. MFI for CLEC-2 increased by 58-fold within the first week and reached a maximum of 1,572-fold increase within the first two weeks of CBMK. Very similar results were obtained on the RNA level. The differential regulation of receptor expression in CBMK was further supported by significant differences in the numbers and types of TF binding sites. P2Y12 and TXA2R, both upregulated only to a low extent in CBMK, probably, are dispensable for megakaryopoiesis. Furthermore, we speculate that CLEC-2 strongly upregulated in early CMBK is important for megakaryopoiesis.

Published
2021
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2. Expression of ADP receptor P2Y12, thromboxane A2 receptor and C-type lectin-like receptor 2 in cord blood-derived megakaryopoiesis

Author

Harald Klüter, Karen Bieback, Stefanie Uhlig, Catharina Gerhards, Foteini Christodoulou, Peter Bugert, and Mani Etemad

Subjects
0301 basic medicine, P2Y receptor, medicine.medical_specialty, Chemistry, Receptor expression, Hematology, General Medicine, 030204 cardiovascular system & hematology, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, Endocrinology, P2Y12, C-type lectin, Internal medicine, medicine, Platelet, Platelet activation, Receptor, Megakaryopoiesis
Abstract

The ADP receptor P2Y12, the thromboxane A2 receptor (TXA2R) and the C-type lectin-like receptor 2 (CLEC-2) mediate platelet activation by different mechanisms. Only little is known about the expres...

Published
2020

3. Expression of ADP receptor P2Y

Author

Catharina, Gerhards, Stefanie, Uhlig, Mani, Etemad, Foteini, Christodoulou, Karen, Bieback, Harald, Klüter, and Peter, Bugert

Subjects
Blood Platelets, Receptors, Purinergic P2, Receptors, Thromboxane, Humans, Lectins, C-Type, Fetal Blood, Platelet Activation, Megakaryocytes, Transcription Factors
Abstract

The ADP receptor P2Y

Published
2020

4. Megakaryocytes and platelets express nicotinic acetylcholine receptors but nicotine does not affect megakaryopoiesis or platelet function

Author

Peter Bugert, Angelika Schedel, Julian Starigk, Dennis Hassmann, Florian Lorenz, Karen Bieback, Anip Sarin, Stefanie Uhlig, and Kerstin Kaiser

Subjects
Adult, Blood Platelets, Male, Nicotine, medicine.medical_specialty, Pilot Projects, Receptors, Nicotinic, 030204 cardiovascular system & hematology, 03 medical and health sciences, 0302 clinical medicine, In vivo, Internal medicine, medicine, Humans, Platelet, Platelet activation, Thrombopoietin, Megakaryocyte Progenitor Cells, Megakaryopoiesis, Chemistry, Cell Differentiation, Hematology, General Medicine, Platelet Activation, Nicotinic agonist, Endocrinology, Case-Control Studies, Female, Megakaryocytes, 030217 neurology & neurosurgery, Ex vivo, medicine.drug
Abstract

In our previous investigations we have shown that platelets and their precursors express nicotinic α7 acetylcholine receptors (nAChRα7) that are involved in platelet function and in vitro differentiation of the megakaryoblastic cell line MEG-01. In this study, we were interested in the expression analysis of additional nAChR and the effects of nicotine in an ex vivo model using megakaryocytic cells differentiated from cord blood derived CD34(+) cells (CBMK) and an in vivo model using blood samples from smokers. CBMK were differentiated with thrombopoietin (TPO) for up to 17 days. Quantitative real-time PCR (QRT-PCR), Western blot analysis and flow cytometry were used to investigate nAChR expression (nAChRα7, nAChRα4, nAChRβ2) and nicotine effects. In blood samples of 15 nonsmokers and 16 smokers platelet parameters (count, mean platelet volume--MPV and platelet distribution width--PDW) were determined as indicators for changes of in vivo megakaryopoiesis. Platelet function was determined by the use of whole blood aggregometry and flow cytometry. The functional role of nAChR was evaluated using specific antagonists in aggregometry. CHRNA7, CHRNA4 and CHRNB2 gene transcripts and the corresponding proteins could be identified in CBMK during all stages of differentiation. Platelets contain nAChRα7 and nAChRβ2 but not nAChRα4. Nicotine had no effect on TPO-induced differentiation of CBMK. There was no significant difference in all platelet parameters of the smokers compared to the nonsmokers. In line with this, cholinergic gene transcripts as well as the encoded proteins were equally expressed in both the study groups. Despite our observation of nAChR expression in megakaryopoiesis and platelets, we were not able to detect effects of nicotine in our ex vivo and in vivo models. Thus, the functional role of the nAChR in these cells remains open.

Published
2015

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