Department of Biochemistry

The Department of Biochemistry has a long history in studying the composition and function of low- and high-molecular weight substances contained in the blood. Through the years, our research advanced to the study of these components under influence of pathophysiological processes such as oncohematological diseases, hemostasis disorders, and thrombosis, including cardiovascular diseases. We investigate the relationships between the individual components of the coagulation cascade, from monitoring the activities of serine proteases and the structure of the resulting fibrin network, to fibrinolysis. We detect acquired and inherited mutations of fibrinogen and look for a relationship between the changed structure of fibrinogen and its properties. For our research, we methodically use new approaches in the form of a combination of liquid chromatography with mass spectrometry (LC-MS/MS) for metabolomics and proteomics. We also use 3D imaging, real-time interaction tracking, and theoretical methods of structural biology, such as molecular modeling and dynamics. Our work aims to clarify the physiological and pathophysiological processes in selected diseases and the use of knowledge in the diagnosis or monitoring of the treatment. Our main areas of research are:

Hemocoagulation and Fibrinolysis

This project aims to describe the relationships between the individual components of the coagulation cascade and their influence by pathophysiological conditions (e.g. oxidative stress). We study blood coagulation from the activation of serine proteases, through the dynamics of formations and structure of the resulting fibrin network, to fibrinolysis. We use not only usual coagulation methods but also new approaches in the form of mass spectrometry, electron microscopy, or methods of structural biology. The acquired knowledge is applied in the development of new diagnostics approaches in coagulopathies (e.g. deep vein thrombosis, pulmonary embolism, myocardial infarction, thrombotic complications in COVID-19).

Characterization of Fibrinogen Disorders

We examine all exons of genes encoding fibrinogen (FGA, FGB, and FGG) in patients with suspected dysfibrinogenemia or hypofibrinogenemia from all over the Czech Republic.  If the mutation is confirmed, we investigate its effect on the structure and function of fibrinogen with specialized methods (fibrin polymerization curves, fibrinolysis, quantification of fibrinopeptide cleavage by HPLC, SEM, LC-MS/MS, structural modeling). Over 100 cases from more than 70 families have been identified at IHBT, of which 16 mutations are newly discovered.

Computer Modeling

We use theoretical methods of structural biology, such as molecular modeling and molecular dynamics, primarily to study fibrinogen and its interactions with other molecules in the blood. Specifically, a study of the effect of post-translational modifications and mutations on the structure and function of fibrinogen, a description of fibrinogen interactions with thrombin and hemostatic snake venoms, and a prediction of the structure of fibrinogen parts that could not be determined experimentally. We also use methods of sequential and phylogenetics analysis.

Metabolomics of Plasma/Serum/Cell extracts in oncohematological and other diseases

We deal with targeted analysis of low molecular weight substances using a combination of liquid chromatography with tandem mass spectrometry (LC-MS/MS). Specifically, we study changes in concentrations of metabolites of glycolysis, citrate cycle, energy pool, carnitine metabolism, tryptophan, and selected amino acids. We found differences in patients with acute myeloid leukemia (AML) depending on the course of the disease. We also monitor oxidative stress markers (malondialdehyde, uric acid, allantoin, etc.) We use spectrophotometry for the determination of carbonylation of proteins.

Protein-protein Interaction by Plasmon Surface Resonation (SPR)

This work aims to design a SPR diagnostic method and protein chip for more accurate stratification of subgroups of oncohematological or other diseases. We designed the protein (micro) chip that analyzes the interaction between proteins associated with the pathogenesis of a given disease and the plasmatic proteome of patients. Another designated Hsp70 trap (micro) chip uses five different subcellular chaperons of the Hsp70 family and is used to detect a damaged subproteome of patients. Protein (micro) chip has already demonstrated the ability to distinguish between different subgroups of myelodysplastic syndrome (MDS) and healthy donors. Whereas Hps70 trap (micro) chip showed the ability to distinguish between MDS patients with low- and high-risk patients. Identification of interacting proteins by mass spectrometry has revealed other potential candidates for increasing the diagnostic sensitivity of the protein chip.

Proteomics of Blood Cells and Other Blood Components

This work aims to clarify physiological and pathophysiological processes in selected diseases or to use knowledge in the diagnosis or monitoring. The main focus is on oncohematological diseases (e.g. MDS) but also on other diseases (e.g. cardiovascular diseases). Another area of research is to monitor the interaction of artificial (polymeric) surfaces with blood. The knowledge is used in a wide range of areas from the design of diagnostic tools to biomedicine (tissue, engineering, implants, etc.).

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