New experimental screening protocols to understand the mechanisms behind thrombosis
08.04.2021
The study entitled Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen is the result of the collaboration between the Quantitative Proteomics Laboratory of Prof. Ruedi Aebersold (ETH, Zurich, Switzerland) and the Laboratory of Protein Chemistry and Molecular Hematology of Prof. Vincenzo De Filippis (University of Padua). Their finding has created a new experimental screening protocol to understand the mechanisms behind thrombosis.
The findings were published in “Nature Communication" and are considered the first step in designing and synthesizing new protease inhibitors with potential drug related applications for several pathologies, including thrombosis. Soon, a blood test or saliva sample will be enough to screen the protease profile with a single test to see if a subjects protease are working correctly, or if one or more show abnormal activity that can cause the disorder.
The biochemical mechanisms determining the physiological coagulation of blood, which prevents blood loss, can occur following vessel (artery or vein) damage caused, for example, by trauma. Blood clot formation, which mainly consists of fibrin and platelets that temporarily “plugs” the damaged vessel. Other processes then trigger how the body will repair the damaged vasculature. It is not clear why, but some clots generate without the onset of a vessel lesion, yet they still cause blood loss. Under these conditions, the formation of a blood clot can obstruct the blood vessel and prevent blood from "downstream" and adequately nourishing tissue, thus leading to the onset of thrombotic disorders such as a heart attack or stroke.
Physiological and pathological conditions that form a blood clot are not easily generated, as a sequence (like a cascade of events) of about twenty proteins are involved and as well as substrates, which are enzyme proteases capable of "breaking down" other proteins. These proteins then become active proteases thus breaking down and activating other proteins, until they generate a clot. There is therefore a sequence of events, which takes the name of "coagulation cascade."
This work has developed a new experimental screening protocol to characterize the substrate specificity of the proteases of the coagulation cascade, i.e. the identification of the specific amino acid sequence that is "broken down" by the various proteases involved in the blood coagulation process.
The focus of the study is on the characterization of a dozen proteases of the coagulation cascade that, under different conditions, generate more than 100,000 unique fragments. Developing this approach is essential to understand the mechanisms of thrombotic disorders, with the goal of creating new drug treatments. Currently, these disorders represent the major cause of death and hospitalization in the industrialized world.
Additionally, the identification of substrates can represent the first step in the design and synthesis of new protease inhibitors that hold potential pharmacological applications in many of the aforementioned disorders. The novel approach is the possibility of using advanced mass spectrometry techniques to identify, not for one, but thousands of substrates for each protease under study within a few hours.
Prof Vincenzo De Filippis explains, “With this in mind, one day we will be able to take a blood or a saliva sample, and conduct a single test to profile the proteases to know if they are working correctly or if one or more show abnormal activity, which can cause disease. This profile will be essential in identifying risk factors for serious illness (e.g. thrombotic disorders) in advance and offering the best available care for each patient using personalized therapeutic approaches.
