Molecular Mechanisms of Recognition of Coagulation Factors (Meccanismi Molecolari di Riconoscimento dei Fattori della Coagulazione)

The biochemical pathways sustaining living organisms, traditionally regarded as independently organized systems, are actually extensively connected by non-canonical protein interactions. The disruption of this finely regulated homeostasis, largely yet to unravel, results in various pathological manifestations. Haemostasis (Chapter 1) is a defence response triggering after vessel walls injuries, which is articulated in the cascade activation of the blood coagulation factors, resulting in the generation of a localized clot. The central reaction of the coagulation cascade is prothrombin activation to mature a-thrombin by FXa, through the generation of the physiologically relevant intermediate prethrombin-2. Active a-thrombin is an ellipsoidal protein, composed of two six-stranded b-barrels encompassing, at their interface, the catalytic triad (His57, Asp102, Ser195). Opposite to the negatively charged active site, two extra positive regions of binding, named exosite I and exosite II, mediate the recognition with several physiologic ligands and substrates. Mature a-thrombin plays a pivotal role in haemostasis, entailing both procoagulant functions (platelets aggregation, fibrin generation) and an anticoagulant one (protein C activation). Beyond coagulation, this serine protease acts at the interface between inflammation, cellular proliferation, and neurodegenerative diseases. The interplay between a-thrombin and proteins traditionally belonging to the central nervous system is undoubtedly a pioneering and yet unexplored topic (Chapter 2.1). When present at high cerebral concentrations a-thrombin triggers a pathologic pro-inflammatory state in the brain, which may be involved in the onset of neurodegenerative diseases. In detail, we refer to as synucleinopathies for a branch of diseases (i.e. Parkinson’s disease) in which intracellular proteinaceous aggregates, mainly composed of a-synuclein, localize both in neurons and in glia. a-synuclein is an abundant presynaptic protein, belonging to the family of naturally unfolded proteins (NUPs), whose physiologic function is still matter of debate. Strikingly, beyond central nervous system, a-synuclein has been detected in plasma and in the haematopoietic lineage, particularly in platelets. Surprisingly, patients suffering from Parkinson’s disease, featuring high a-synuclein concentrations, are characterized by lower ischaemic attacks, due to platelets abnormalities and impaired aggregation. Being these cells the main trigger of a-thrombin, we purposed to investigate the interaction between this enzyme and a-synuclein (Chapter 2.2). Our data clearly demonstrate that the two proteins bind with an affinity physiologically relevant for the concentrated microenvironment surrounding platelets. In the binary complex, a-synuclein interacts promiscuously with a-thrombin exosites by its negatively charged de-structured C-terminus, thus scavenging hyper-aggregation phaenomena. In this intricate network, positive upregulation between coagulation and inflammation has been well established and extensively studied (Chapter 3.1). During sepsis, systematically activated immune response results in an exaggerated and detrimental inflammation, usually coupled to disseminated intravascular coagulation. In this scenario, exogenous proteases may play a relevant role during the early stages of the infection by directly activating coagulation. Several pathogen microorganisms express and secrete subtilisin-like serine proteases (subtilases), characterized by a broad specificity of cleavage. We purposed to investigate the effects of subtilases-catalysed proteolysis of thrombin zymogens prethrombin-2 (Chapter 3.2) and prothrombin (Chapter 3.3) by using the commercially available subtilisin Carlsberg, as a prototype for the superfamily. From the proteolysis and enzymatic assays data, it strikingly emerged that subtilisin activates both the zymogens to a novel thrombin-like specie, by the non-canonical hydrolysis of Ala470(149a)-Asn471(149b) peptide bond. The novel active specie, we named sPre2, is a non-covalent complex featuring the same a-thrombin cleavage specificity, with a catalytic efficiency ≈150-fold inferior to the physiologic enzyme. From fluorescence titrations and surface binding resonance, it emerged that sPre2 is characterized by a fully competent exosite II, an imperfect exosite I and a correctly moulded active site, which however features an impaired mechanism of substrate conversion. Both experimental and clinical evidences clearly demonstrate the crucial importance of maintaining coagulation homeostasis, which, in physiologic conditions, is slightly unbalanced towards a haemorrhagic state to keep blood fluid in the intact vessels. A disruption of this equilibrium due to increase of functioning leads to pathological manifestations generically referred to as thrombosis (Chapter 4.1). Unfortunately, classical anticoagulant therapy presents well-documented limitations and bleeding side effects, driving the continuous efforts to develop new, safer drugs. In the late years, a hot research topic is represented by the engineering of natural anticoagulants from hematophagous organisms. Among all, anticoagulant hirudin from the medicinal leeches is the most popular compound, being a-thrombin most potent and specific natural inhibitor. In this work, we propose a novel strategy for hirudin production in E. coli, by conjugation to SUMO (small ubiquitin-like modifier protein), a eukaryotic chaperon which enhances protein folding and solubility (Chapter 4.2). The so-obtained recombinant hirudin is characterized by the same folding, spectroscopic features, and anti-thrombin activity of the natural variant. In conclusion, coagulation is undoubtedly one of the most articulated and fascinating physio-pathologic systems regulating body homeostasis, displaying several connections with other biochemical pathways. The molecular mechanisms of recognition of the coagulation factors, yielding traditional or original protein interactions, is still a highly unexplored topic