Modulating effect of recombinant lactoferrin isolated from the milk of transgenic goats on platelet aggregation activity

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Abstract

This work showed for the first time that recombinant lactoferrin isolated from the milk of transgenic goats binds to the plasma membrane of platelets. The interaction of recombinant lactoferrin with platelets leads to the potentiation or inhibition of agonist-induced platelet aggregation depending on the type of agonist (adenosine diphosphate, thrombin, plant lectins WGA, Con A, and SNA). Using fluorescently labeled antibody CD42b it was shown by flow cytometry that recombinant lactoferrin binds to GPIb on platelets.

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About the authors

Ekaterina V. Shamova

Belarusian State University

Author for correspondence.
Email: shamova@tut.by

PhD in Biology, Senior Research Scientist, Research Laboratory of Biophysics and Biotechnology, Department of Biophysics, Faculty of Physics

Belarus, 4, Nezavisimosti Ave., 220030, Minsk

Daria V. Grigorieva

Belarusian State University

Email: dargr@tut.by

PhD in Biology, Senior Research Scientist, Research Laboratory of Biophysics and Biotechnology, Department of Biophysics, Faculty of Physics

Belarus, 4, Nezavisimosti Ave., 220030, Minsk

Irina V. Gorudko

Belarusian State University

Email: irinagorudko@gmail.com

PhD in Biology, Associate Professor, Associate Professor of the Department of Biophysics, Faculty of Physics

Russian Federation, 4, Nezavisimosti Ave., 220030, Minsk

Anastasia N. Sveshnikova

Lomonosov Moscow State University; Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences

Email: agolomy@gmail.com

PhD of Physics and Mathematics, Senior research scientist, Department of Biophysics, Faculty of Physics; Head of the Laboratory of Intracellular Signaling and Systems Biology

Russian Federation, 1, Leninskie gory, Moscow, 119991; 4, Kosigina street, Moscow, 119991

References

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2. Fig. 1. Binding of recombinant lactoferrin (rLF) conjugated to a fluorescent label (FITC) with platelets: washed platelets were incubated with rLF-FITC (200 μg / ml) for 10 minutes at +23 ° C, the samples were diluted and analyzed by flow cytometer . At least 10,000 platelets were counted in each sample. The binding of rLF to the platelet membrane was assessed by the number of positive events for platelets carrying rlf labeled on their surface and labeled with FITC

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3. Fig. 2. Influence of recombinant lactoferrin (rLF) on platelet aggregation induced by the maczemic agonists (adenosine diphosphate (ADP), thrombin) and plant lectins (WGA - specific wheat germ lectin, Con A - mannose-binding lectin canavalia, aceta, aus, acetocephalus, aceptotoxicity) black). Platelets were incubated with rLF for 2 minutes, and then an agonist was added. In fig. a, b, d - typical kinetic curves of platelet aggregation induced by ADP (a), thrombin (b) and WGA (g), respectively, in the presence and in the absence of rLF. In fig. c, d, e — dependence of the degree of aggregation (T) (c and d) and the stability parameter of lectin-induced aggregates (R) (e) on the concentration of rLF. Agonist concentrations: ADP - 2.5 μM, thrombin - 12.5 μg / ml, WGA - 5 μg / ml, Con A - 100 μg / ml, SNA - 75 μg / ml. In the case of lectin-induced aggregation, platelet disaggregation was initiated by adding the corresponding haptenic carbohydrates: 100 mM GlcNAc (WGA), 60 mM α-methide-D-mannoside (Con A), 60 mM lactose (SNA). The concentration of rLF on graphs with kinetic curves is given in µg / ml. The arrow indicates the moment of addition of the haptenic carbohydrate.

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4. Fig. 3. Reducing the number of CD42b binding sites with GPIb in the presence of recombinant lactoferrin (rLF). Platelets were incubated with rLF (100–1000 μg / ml) for 10 minutes at +23 ° C, then CD42b R-PE (1% by volume) was added and incubated for another 5 minutes at +23 ° C. The obtained samples were diluted and used for subsequent analysis on a flow cytometer. For each sample, at least 10,000 platelets were analyzed. GPIb exposure on platelet surfaces was judged by the fluorescence intensity of CD42b R-PE antibodies. * p <0.05 compared with samples in the absence of rLF (control)

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Copyright (c) 2018 Shamova E.V., Grigorieva D.V., Gorudko I.V., Sveshnikova A.N.

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