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Coagulation protein factors: discovering novel interactions of thrombin
Full text linkThrombin is a serine protease of the chymotrypsin family. Compared to chymotrypsin, thrombin displays several insertion loops, responsible for the unique substrate specificity of the enzyme. Two different insertions shape and narrow the access to the active site, while the interaction of binders involves allosteric sites, called exosite-I (Anion Binding Exosite-I or Fibrinogen Recognition Site) and exosite-II (Anion Binding Exosite-II or Heparin Binding Site). These contain electropositive amino acid residues and are localized at opposite poles of the active site, representing two potential exosites for the binding of macromolecular ligands. Exosite-I is involved in binding to fibrinogen, platelet receptor PAR-1, thrombomodulin, and to endogenous (i.e. heparin cofactor II) and exogenous (i.e. C-terminal tail of hirudin) inhibitors. Exosite-II interacts with heparin, F2 prothrombin fragment, and physiological inhibitors such us antithrombin III and protease nexin-I. Contrary to chymotrypsin, the proteolytic activity of thrombin is enhanced upon binding of Na+, that stabilizes the enzyme into a more open and rigid conformation.
Thrombin is a multifunctional enzyme that plays a key role at interface between coagulation, inflammation and nervous system. The protease is involved in numerous physiological and pathological processes, including haemostasis and thrombosis, inflammation and chemotaxis, cellular proliferation and tumor growth, angiogenesis and neurodegenerative diseases, manifesting pleiotropic effects. For example, low concentrations of thrombin (i.e. 1-10nM) can influence glia cell mitosis and neuronal out-growth, acts as mitogen. Conversely, higher concentration of the enzyme (100nM) has been shown to induce apoptosis in motor neurons and to determine in the brain a pro-inflammatory state. Instead, in vivo, the dynamic concentration of free thrombin during coagulation cascade reactions is estimated to vary from 1nM to over 100 – 500nM. Typically low concentration are associated with platelet activation and loosely organised fibrin strands susceptible to fibrinolysis; higher concentration produce tightly packet fibrin strands capable of forming a stable clot. Some of these effects are mediated by activation of Protease Activated Receptors (PARs). The general mechanism by which proteases activate PARs is the same: enzymes cleave at specific sites within the extracellular amino terminus of the receptors; this cleavage exposes a new amino terminus that serves as a tethered ligand domain, which binds to conserved regions in the second extracellular loop of the cleaved receptor, resulting in the initiation of signal transduction. All these observations argue in favor of a biochemical communication between the different mechanisms regulating the cellular effects of thrombin.
The general aim of my PhD project was to identify novel effectors of thrombin, whose interaction may have important implications in defining the biochemical processes that regulate the onset and progression of cardiovascular diseases, neurodegenerative and autoimmune diseases.
During the first year, I studied the effect of beta2-glycoprotein I (b2GPI) on the procoagulant (i.e. fibrin generation and platelet aggregation) and anticoagulant (i.e. generation of activated protein C) functions of thrombin (Chapter 2). b2GPI, identified as the major antigen of antiphospholipid syndrome (APS), functions as a physiologic anticoagulant by inhibiting the key procoagulant activities of the protease, without affecting its unique anticoagulant function. Our experiments, conducted by surface plasmon resonance (SPR), clarify the binding mode of interaction: b2GPI binds to thrombin exosites, while the active site remains free and accessible for substrate binding.
In the second year of my PhD course, I have investigated the interaction between alpha-synuclein (a-Syn) and human thrombin (Chapter 3). a-Syn is a small soluble presynaptic protein implicated in different neurodegenerative disorders. Recent studies indicated that a-Syn is able to inhibit platelets degranulation, upon thrombin stimulation. In addition, clinical studies indicated that the incidence of ischemic stroke in patients with Parkinson disease is lower than in controls, and platelet aggregation is also significantly decreased. Our results suggest that the acidic C-terminal portion of -Syn binds to thrombin exosites (Kd ~ uM). Consequently, we speculate that the complex [a-Syn – thrombin] effectively hinders platelet aggregation, due to the interaction of the N-terminal domain on the platelets surfaces.
During the last year, I studied human ceruloplasmin (CP) as a possible binder of thrombin (Chapter 4). The plasma level of CP is an important diagnostic indicator of inflammatory disease, such as Rheumatoid Arthritis (RA), a chronic systemic inflammatory autoimmune disorder. As observed with CP, thrombin concentration is markedly increased in inflamed tissues and specifically in the synovial fluid of RA patients. We conclude that the anti-inflammatory function of CP is regulated by thrombin: the enzyme, in fact, proteolytically hinders the antioxidant activity of CP. These results are confirmed in RA patients treated with hirudin that have clinical symptoms ameliorated. These data are unprecedented and set the basis for elucidating the biochemical mechanisms underlying the progression of inflammation in RA patients
Understanding thrombin allostery: Effect of exosite-1 and exosite-2 binders on enzyme recognition and catalysis
No full textIdentification of methionine sulfoxide at position 1606 of von Willebrand Factor (vWF) as a risk factor for thrombosis in patients with Antiphospholipid Syndrome (APS).
No full textThrombin allostery: effect of exosite-1 and exosite-2 binders on the enzyme molecular recognition and catalysis
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Thrombin binds to human ceruloplasmin and proteolytically hinders its antioxidant activity
No full textThrombin binds to human ceruloplasmin and proteolytically hinders its antioxidant activity
No full textNoncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction
Full text linkThe advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin
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