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The fibrinogen molecule contains multiple binding motifs for different types of cellular receptors, acting as a molecular link between coagulation and cell adhesion. In this study we generated a truncated form of the fibrinogen molecule lacking the Bβ1-42 sequence by site-specific proteolysis and evaluated the role of the fragment in adhesive capabilities of platelets, endothelial and cancer cells. Fibrinogen with the removed Bβ1-42 sequence and fibrin without the Bβ15-42 fragment (desβ1-42 fibrinogen and desABβ15-42 fibrin) were obtained by proteolysis using the specific protease from the venom of Echis multisquamatis. The cleaved fragment was purified by HPLC and was identified using MALDI-TOF. ADP- and collagen-induced aggregation of washed platelets in the presence of fibrinogen desBβ1-42 was studied using an aggregometer. Proliferation of mice aortic endothelial cells (MAEC) and human umbilical vein endothelial cells (HUVEC) was studied using the fibrin desABβ15-42 as the scaffold. Cell viability was quantified by the MTT test (MAEC). Generation time was calculated for the estimation of proliferative activity of HUVEC. Lung cancer cell line Н1299 was used to evaluate cancer cell motility in vitro using the scratch assay. Direct comparison of cellular behavior in the presence of truncated vs native forms demonstrated attenuated cell adhesion in the presence of fibrinogen desBβ1-42 and fibrin desBβ15-42. The platelet aggregation rate was only slightly decreased in the presence of fibrinogen desBβ1-42 but resulted in 15-20% disaggregation of adhered platelets. We also observed the substantial decrease of generation time of HUVEC and inhibition of viability of MAEC cells grown on scaffolds of a desABβ15-42 matrix. Finally, desBβ1-42 modulated the motility of H1299 cells in vitro and suppressed the wound healing by 20% compared to the full-length fibrinogen. We postulate that fragment 1-42 of the BβN-domain of fibrinogen is not sufficient for platelet aggregation, however it may contribute to platelet clot formation in later stages. At the same time, this fragment may be important for establishing proper cell-to-cell contacts and cell viability of endothelial cells. Also, 1-42 amino acid fragment of the BβN-domain supported the migration of cancer cells suggesting that interactions of fibrinogen with cancer cells could be a target for anticancer therapy. The Bβ1-42 fragment of fibrinogen contributes to efficient intracellular interactions of different types of cells, including platelets, endothelial cells and cancer cells.

6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is a key regulatory enzyme of glucose metabolism. This bifunctional enzyme controls glycolysis and gluconeogenesis as well as glucose phosphorylation and metabolism. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) plays significant role in the glycolysis regulation in the heart as well as in some other organs (kidney, brain and lung). Results of this investigation clearly demonstrated that in the glioma cells as well as in some mouse organs are presented several new unique alternative splice variants of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2. These PFKFB-2 splice variants are a result of deletions or/and inserts in catalytic regions of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase as well as in С-terminal regulatory region. Alternative splice variants of PFKFB-2 from glioma and mouse cells contain different inserts after 2nd exon but mouse splice variant has also deletion in 6-phosphofructo-2-kinase region and insert in C-terminus. Another splice variants of mouse PFKFB-2 have deletions in 6-phosphofructo-2-kinase or fructose-2,6-bisphosphatase regions, since one splice variant has deletion of all catalytic domains. In conclusion, this study provides evidences that alternative splicing of PFKFB-2 mRNA possibly has a significant role in glycolysis regulation via creation of monofunctional isoforms of this enzyme.

A bifunctional PFKFB enzyme is a key regulator of glycolysis. Four different genes encode the synthesis of multiple PFKFB isoforms. We analyzed data concerning structural organization of PFKFB genes and alternative splice variants of PFKFB mRNA, as well as expression and molecular mechanisms of regulation of different PFKFB isoforms in malignant tumors and at hypoxia, and mechanisms of hormonal regulation. Hypoxia inducible factor (HIF)-dependent mechanisms of regulation of the expression of different PFKFB variants are presented and discussed.