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• W1488896445 abstract "Human platelets contain several clotting factors including fibrinogen that are stored in the α-granules. Some of these clotting factors are synthesized by megakaryocytes, such as von Willebrand factor (VWF), while others are taken up from plasma. The origin of platelet fibrinogen was controversially debated between 1985 and 1995. Most data demonstrated that platelet fibrinogen have its origin exclusively in plasma (Harrison et al, 1989; Handagama et al, 1993), although endogenous synthesis of fibrinogen during megakaryocyte development has also been reported (Belloc et al, 1985). The development of microarray technology has enabled semi-quantitative analysis of the expression levels of thousands of genes in parallel. Applying this technique to platelets, fibrinogen α-chain (FGA) mRNA has been detected in higher abundance than VWF mRNA. Although fibrinogen β-chain (FGB) and fibrinogen γ-chain (FGC) were also present on the array, results for the corresponding platelet messages were found to be below the detection limit of the assay (Coppinger et al, 2004). In contrast, negative results for all fibrinogen messages have been shown by gene expression analysis of CD34-derived megakaryocytic cells (Tenedini et al, 2004). Considering these conflicting results obtained by microarray analysis, we investigated the existence of fibrinogen gene transcripts in human platelets using a more sensitive method, quantitative real-time polymerase chain reaction (PCR). Quantification of all fibrinogen messages in parallel in human platelets has not been performed before. Platelet isolation, mRNA extraction and quantification of platelet VWF were performed as described by Rox et al (2004). Platelet numbers before RNA isolation ranged from 2 to 5 × 109/l corresponding to 4–10 × 108 platelets per PCR. The sequences of primers and probes for detection of FGA, FGB and FGC mRNA are given in Table I. PCRs were performed separately for each fibrinogen gene in duplicates in a 20 μl volume using the Quantitect reverse transcription-PCR Kit (Qiagen). Thermal cycling and real-time detection of amplification was performed using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems) and the following amplification profile: 50°C for 30 min, 95°C for 15 min followed by 40 cycles of 94°C for 15 s and 60°C for 30 s. RNA calibrator sequences were made by in vitro transcription as previously described by Rox et al (2004). The detection limit of each real-time PCR assay was below 10 copies per reaction as determined by limiting dilution series. Analysis of platelets from eight healthy volunteers by RT-PCR showed that FGA mRNA, FGB mRNA and FGC mRNA were absent. All samples contained VWF messages (range: 2200–9500 copies per PCR, corresponding to 10–40 transcripts/106 platelets) confirming the platelet origin of the samples. Our findings did not indicate expression of fibrinogen genes in human megakaryocytes/platelets, confirming the well-known plasmatic origin of platelet fibrinogen. Unspecific hybridization or cross-hybridization to homologue sequences may have contributed to the positive result for platelet FGA transcripts by microarray analysis. This example shows that microarray results need to be validated by independent mRNA quantification techniques and that the exponentially increasing gene expression data has to be interpreted with caution." @default.
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• W1488896445 date "2005-07-20" @default.
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• W1488896445 title "Absence of fibrinogen alpha-, beta- and gamma-chain mRNA in human platelets" @default.
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• W1488896445 doi "https://doi.org/10.1111/j.1365-2141.2005.05654.x" @default.
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