Evaluation of Platelet Functional Activity in Healthy BALB/c Mice and in EMT-6 Breast Cancer Orthotopic Model

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Abstract

Platelets, anuclear cell fragments circulating in the blood, can be considered as vascular wall and latent thrombosis sensors. However, the heterogeneity of normal platelet indicators is too large for the correct «reading» of information from these sensors. Therefore, it is necessary to fine-tune methods for assessing the functional state of mouse platelets in order to then check under controlled conditions how various pathological processes change the state of platelets. In this work, we adapted previously developed flow cytometry platelet functional activity tests for use in blood samples from BALB/c mice and evaluated changes in platelet status in an in vivo model of spontaneous metastatic breast cancer EMT-6. Continuous and endpoint flow cytometry were used. Platelet shape change, decreased GPIb antibody binding, P-selectin exposure, integrin activation, calcium mobilization, and phosphatidylserine exposure were observed. Several functional activity differences between human and murine platelets were obtained. First, according to all observed parameters, activation of mouse platelets in response to 2.5 µm of ADP was significantly weaker than activation in response to 200 µm of AYPGKF (PAR4 thrombin receptor agonist). Second, reversible activation of integrins and shape change were observed in response to ADP. The main difference between platelets of mice with a tumor was a significant decrease in their number when isolated from the blood. Additionally, some responses were diminished: binding of antibodies to GPIb, shape change, activation of integrins, and procoagulant response. The results indicate that a tumor in the terminal stage probably reduces the number of circulating platelets and causes their damage.

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About the authors

A. A. Mishukov

Center for Theoretical Problems of Physicochemical Pharmacology of the RAS

Email: asve6nikova@yandex.ru
Russian Federation, Moscow

S. Gaur

Moscow Institute of Physics and Technology

Email: asve6nikova@yandex.ru
Russian Federation, Dolgoprudny

E.-I. A. Adamanskaya

Center for Theoretical Problems of Physicochemical Pharmacology of the RAS; Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology, and Immunology

Email: asve6nikova@yandex.ru
Russian Federation, Moscow; Moscow

M. A. Panteleev

Center for Theoretical Problems of Physicochemical Pharmacology of the RAS; Moscow Institute of Physics and Technology; Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology, and Immunology

Email: asve6nikova@yandex.ru
Russian Federation, Moscow; Dolgoprudny; Moscow

A. N. Sveshnikova

Center for Theoretical Problems of Physicochemical Pharmacology of the RAS; Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology, and Immunology

Author for correspondence.
Email: asve6nikova@yandex.ru
Russian Federation, Moscow; Moscow

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2. Fig. 1. Overall characteristics of the orthotopic in vivo model of EMT-6 breast cancer: (a) - EMT-6 tumour growth in the experiments performed, mean values ± SD; Comparison of final tumour mass (b), final tumour volume (c) and number of superficial lung metastases in mice (d) in the two experiments performed, Student's t-test with Welch's correction, ns - p > 0.05, * - p < 0.05; (e) - kinetics of change in mouse weight normalised to day 0 and tumour size, mean values from two experiments ± standard deviation; (f) - kinetics of change in pain/distress index and tumour growth rate, mean values ± standard deviation.

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3. Fig. 2. Results of end-point cytometry of platelets from healthy and tumour-bearing EMT-6 mice: (a) - concentration of platelets isolated from whole blood of healthy and tumour-bearing mice, Welch's parametric test, p < 0.001; (b) - median direct light scattering as a parameter of platelet size, Welch's parametric test, p < 0. 0001; P-selectin exposure (c), decreased actin binding to GPIb (d) and increased percentage of annexin V-positive platelets (e) upon platelet activation by 2.5 μM ADP, a combination of 2.5 μM ADP and 250 μg/ml fucoidan, 200 μM AYPGKF or 40 nM thrombin. Two-factor analysis of variance with Sidak's multiple comparisons, ns - p > 0.05, ** - p < 0.01, **** - p < 0.001.

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4. Fig. 3. Results of continuous flow cytometry of platelets. Changes in free Ca2+ ion levels (a), fibrinogen binding (b) and platelet shape change (c) upon activation (using 0.5 μM ADP as an example), mean values ± standard deviation; (d) - free calcium levels in resting platelets, Welch's parametric test, * - p < 0. 05. Calcium mobilisation (e), fibrinogen binding (f) and shape change (g) upon activation of platelets from healthy mice (white) and EMT-6 tumour-bearing mice (grey) by ADP (0.5 and 2.5 μM), AYPGKF (200 μM), fucoidan sequence (250 μg/ml) and ADP (2.5 μM), and thrombin (40 nM). Two-factor analysis of variance with Sidak's multiple comparisons, ns - p > 0.05, ** - p < 0.01, *** and **** - p < 0.001.

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