In addition to a major role in hemostasis and thrombosis, platelets have many other functions, including modulation of the immune response. Activated platelets release a number of potent chemokines, such as NAP-2 (CXCL7), RANTES (CCL5), and platelet factor 4 (PF4; CXCL4). PF4 is stored in platelet α-granules at very high concentrations and is released upon cell activation at the sites of inflammation and injury. PF4 is known as a key player in heparin-induced thrombocytopenia, a severe autoimmune complication of heparin therapy, but the roles of PF4 in other immunogenic pathologies are not well understood. In this study, we evaluated the direct effects of PF4 on human T-lymphocytes in vitro with respect to apoptosis.

T-cells were immunomagnetically isolated from the blood of healthy donors and cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, antibiotics (penicillin and streptomycin) and L-glutamine during 24 hours in the absence or presence of recombinant wild-type human PF4. Aliquots were taken after 2, 4, 6, 12 and 24 hours of cultivation and the cells were analyzed for ultrastructural and biochemical signs of apoptosis using transmission electron microscopy, flow cytometry, and Western blot analysis.

The vast majority of control T-lymphocytes had a morphology corresponding to time-dependent changes characteristic of different stages of apoptosis. At the early stages (up to 6 hours) the cells lost microvilli from the plasma membrane. At the later stages deep invagination of nuclear and plasma membranes as well as chromatin condensation were observed. The plasma membrane formed blebs and a lot of fragments of dead cells (apoptotic bodies) were revealed. The mitochondria, endoplasmic reticulum and Golgi apparatus were clearly visible in the cytoplasm.

The addition of PF4 to the cells at a final concentration of 2 μg/ml reduced the degree of apoptotic changes. Many T-cells still developed the morphological changes associated with apoptotic cells; however most of them did not progress past the described early stages of apoptosis, even up to 24 hours. These cells maintained a regular oval shape, but the nuclear membrane formed invaginations and chromatin condensed and relocated to the nuclear periphery. The apoptotic bodies were less frequent than in control. The mitochondria, endoplasmic reticulum, vacuoles and lysosomes were well distinguished in the cytoplasm but their number was decreased. However, there was also a subpopulation of T-cells that had an oval shape, the nucleus and plasma membrane were without invaginations, the nucleus was roundish with diffuse chromatin and the nucleolus was clearly seen in the nucleus. The morphology of this subpopulation corresponds to proliferated cells.

In the presence of PF4 added at a final concentration of 100 μg/ml most of the cells kept the typical morphology of proliferating cells up to 24 hours. The plasma membrane had a regular round shape, it was smooth and without invaginations. The nucleus usually occupied a large part of the cell; it was rounded and located centrally. The nucleolus was clearly visualized in the nucleus and the nuclear substance was distributed homogeneously. The ribosomes, the Golgi apparatus and the endoplasmic reticulum were well distinguished in the cytoplasm. The mitochondria had a regular shape with well-differentiated cristae. Most often, accumulation of the mitochondria was observed medially, near the nucleus.

The observed morphological changes were correlated with expression of molecular markers and other signs of apoptosis. Bcl-2 and caspase-3 were not expressed in the T-cells treated with PF4 at a concentration of 100 µg/ml, while caspase-3 was detected in the control cells and in the cells treated with PF4 at a concentration of 2 µg/ml after 4 and 6 hours of incubation. Consistent results were obtained with assessment of the degree of apoptosis by Annexin V binding and propidium iodide staining.

In conclusion, our results provide direct evidence for dose-dependent anti-apoptotic effects of PF4 on T-cells, suggesting that PF4 may sustain an immune response by extending T-lymphocyte survival.

(Research supported by NIH grant HL110860 and the Program of Competitive Growth of Kazan Federal University)

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.

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