Coagulation protein factors: discovering novel interactions of thrombin

Thrombin 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

Non-canonical proteolytic activation of human prothrombin by subtilisin from Bacillus subtilis may shift the procoagulant\ue2\u80\u93anticoagulant equilibrium toward thrombosis

Blood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)-mediated conversion of the prothrombin (ProT) zymogen to active -thrombin (T). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg-271, generating the inactive precursor pre-thrombin-2 (Pre2), which is further attacked at Arg-320 \u2013Ile-321 to yield mature T. Whereas the mechanism of physiological ProT activation has been elucidated in great detail, little is known about the role of bacterial proteases, possibly released in the bloodstream during infection, in inducing blood coagulation by direct proteolytic ProT activation. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies. Here, we show that addition of subtilisin (50 nM to 2 M), a serine protease secreted by the non-pathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active Pre2, a nicked Pre2 derivative with a single cleaved Ala-470 \u2013Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470 \u2013Asn-471 is instrumental for the onset of catalysis in Pre2, which was, however, reduced about 100 \u2013200-fold compared with T. Of note, Pre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of ProT by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant\u2013anticoagulant equilibrium toward thrombosis

Interplay between conformational selection and zymogen activation

Trypsin-like proteases are synthesized as zymogens and activated through a mechanism that folds the active site for efficient binding and catalysis. Ligand binding to the active site is therefore a valuable source of information on the changes that accompany zymogen activation. Using the physiologically relevant transition of the clotting zymogen prothrombin to the mature protease thrombin, we show that the mechanism of ligand recognition follows selection within a pre-existing ensemble of conformations with the active site accessible (E) or inaccessible (E) to binding. Prothrombin exists mainly in the Econformational ensemble and conversion to thrombin produces two dominant changes: a progressive shift toward the E conformational ensemble triggered by removal of the auxiliary domains upon cleavage at R271 and a drastic drop of the rate of ligand dissociation from the active site triggered by cleavage at R320. Together, these effects produce a significant (700-fold) increase in binding affinity. Limited proteolysis reveals how the E-E equilibrium shifts during prothrombin activation and influences exposure of the sites of cleavage at R271 and R320. These new findings on the molecular underpinnings of prothrombin activation are relevant to other zymogens with modular assembly involved in blood coagulation, complement and fibrinolysis

Identifying conformational changes with site-directed spin labeling reveals that the GTPase domain of HydF is a molecular switch

[FeFe]-hydrogenases catalyse the reduction of protons to hydrogen at a complex 2Fe[4Fe4S] center called H-cluster. The assembly of this active site is a multistep process involving three proteins, HydE, HydF and HydG. According to the current models, HydF has the key double role of scaffold, upon which the final H-cluster precursor is assembled, and carrier to transfer it to the target hydrogenase. The X-ray structure of HydF indicates that the protein is a homodimer with both monomers carrying two functional domains: a C-terminal FeS cluster-binding domain, where the precursor is assembled, and a N-terminal GTPase domain, whose exact contribution to cluster biogenesis and hydrogenase activation is still elusive. We previously obtained several hints suggesting that the binding of GTP to HydF could be involved in the interactions of this scaffold protein with the other maturases and with the hydrogenase itself. In this work, by means of site directed spin labeling coupled to EPR/PELDOR spectroscopy, we explored the conformational changes induced in a recombinant HydF protein by GTP binding, and provide the first clue that the HydF GTPase domain could be involved in the H-cluster assembly working as a molecular switch similarly to other known small GTPases

Polyphenols as Potential Therapeutic Drugs in Neurodegeneration

Several therapeutic approaches have been suggested so far for the treatment of neurodegenerative diseases, but to date, there are no approved therapies. The available ones are only symptomatic; they are employed to mitigate the disease manifestations and to improve the patient life quality. These diseases are characterized by the accumulation and aggregation of misfolded proteins in the nervous system, with different specific hallmarks. The onset mechanisms are not completely elucidated. Some promising approaches are focused on the inhibition of the amyloid aggregation of the proteins involved in the etiopathology of the disease, such as Aβ peptide, Tau, and α-synuclein, or on the increase of their clearance in order to avoid their aberrant accumulation. Here, we summarize traditional and new therapeutic approaches proposed for Alzheimer’s and Parkinson’s diseases and the recent technologies for brain delivery

Coagulation protein factors: discovering novel interactions of thrombin

Thrombin 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.La trombina è una proteasi serinica appartenente, per omologia di sequenza, alla famiglia della chimotripsina dalla quale differisce per la presenza di numerosi loop d’inserzione, che le conferiscono una peculiare specificità di substrato. Essa si presenta come un ellissoide caratterizzato da due beta-barrels, alla giunzione dei quali si colloca la cavità che ospita il sito attivo. Il riconoscimento molecolare dei diversi effettori, invece, è mediato da due regioni superficiali elettropositive, diametralmente opposte e circondanti la cavità catalitica. Queste sono definite, rispettivamente, esosito-I (Anion Binding Exosite-I o Fibrinogen Recognition Site) ed esosito-II (Anion Binding Exosite-II o Heparin Binding Site). L’esosito-I è coinvolto nel legame della trombina al fibrinogeno, al recettore piastrinico PAR-1, alla trombomodulina, e a inibitori endogeni, come il fattore eparinico II, ed esogeni come la coda C-terminale dell’irudina. L’esosito-II rappresenta il sito di legame per l’eparina, per il frammento F2 della pro-trombina e per inibitori fisiologici come l’antitrombina III e la nexina-I. A differenza della chimotrispsina, l’attività proteolitica della trombina è aumentata dal binding del Na+ che stabilizza l’enzima in una conformazione più aperta e rigida. La trombina è una proteasi multifunzionale: da una parte gioca un ruolo importante nella cascata coagulativa, dall’altra interviene in modo fondamentale nei processi infiammatori a carico del sistema nervoso centrale. Infatti, la trombina svolge un ruolo chiave all’interfaccia tra coagulazione, infiammazione, differenziamento cellulare, angiogenesi e malattie neurodegenartive, manifestando così effetti pleiotropici. Studi in vitro hanno evidenziato come tale proteina sia in grado di modulare la permeabilità vascolare, la formazione di neo-vasi e la ritrazione di neuriti su cellule di neuroblastoma; per di più sembra svolgere attività mitogena a carico di cellule muscolari ed endoteliali. Questi effetti si realizzano a basse concentrazioni (1-10nM), mentre concentrazioni maggiori (100nM) sembrano essere nocive e pro-infiammatorie a livello cerebrale. Allo stesso modo, elevate concentrazioni plasmatiche di trombina (100-500nM) portano alla formazione di un clot compatto di fibrina, non suscettibile a fibrinolisi. Alcuni studi hanno dimostrato come la maggior parte delle funzioni non emostatiche si manifestino mediante l’attivazione dei recettori piastrinici PAR (Protease Activated Receptors), recettori transmembrana accoppiati a proteine-G. Nel dettaglio, il dominio extracellulare del PAR-1, in seguito a proteolisi promossa dalla trombina, interagisce col corpo recettoriale favorendo la trasduzione del segnale all’interno di piastrine e macrofagi. Il tutto si traduce in una risposta pro-aggregante e pro-infiammatoria. Questi dati suggeriscono la presenza di una stretta comunicazione biochimica tra i vari meccanismi che regolano i differenti effetti cellulari della trombina. Alla luce di queste considerazioni, l’obiettivo saliente del mio Progetto di Dottorato è stato quello di identificare nuovi effettori della trombina, i cui meccanismi di interazione possono avere importanti ricadute nella definizione dei processi biochimici che regolano l’insorgenza e la progressione delle malattie cardiovascolari, neurodegenerative ed autoimmuni. Durante il primo anno ho studiato l’effetto della beta2 glicoproteina I (b2GpI) sulle funzioni pro- e anti- coagulanti della trombina (Capitolo 2). La b2GpI, identificata come il principale antigene della sindrome da anticorpi antifosfolipidi (APS), è in grado di inibire le attività procoagulanti (generazione di fibrina ed aggregazione piastrinica) della trombina in vitro, senza compromettere l’unica sua funzione anticoagulante, ovvero la generazione di Proteina C attiva. I nostri esperimenti, condotti principalmente mediante surface plasmon resonance (SPR) hanno permesso inoltre di chiarire il binding mode di interazione delle due proteine: la b2GpI si lega agli esositi della trombina, il cui sito attivo rimane quindi accessibile al substrato. Nel corso del secondo anno ho indagato l’interazione tra l’alpha-sinucleina (a-Sin) e la trombina umana (Capitolo 3). a-Sin è una piccola proteina solubile presinaptica, implicata in diverse patologie neurodegenerative. Recentemente è stato dimostrato come l’a-Sin sia in grado di inibire l’attivazione e quindi l’aggregazione delle piastrine quando stimolate da trombina, limitando il rilascio degli alpha-granuli. Inoltre pazienti affetti dal morbo di Parkinson sono meno soggetti ad attacchi ischemici e presentano una velocità di aggregazione piastrinica significativamente ridotta. I risultati da noi ottenuti indicano che la porzione acida C-terminale dell’a-Sin è in grado di legarsi alla trombina con un’affinità nell’ordine del basso micromolare, coinvolgendo i due esositi. Quindi, il complesso [a-Sin - trombina] ostacola efficacemente l’aggregazione piastrinica, molto probabilmente in seguito all’ancoraggio del dominio N-terminale sulla superficie delle piastrine. Infine, durante l’ultimo anno, è stata presa in considerazione la ceruloplasmina umana (CP), quale possibile binder della trombina (Capitolo 4). Elevati livelli di CP sono stati individuati in pazienti affetti da artrite reumatoide, malattia infiammatoria cronica autoimmunitaria a carico delle articolazioni sinoviali. Come osservato per la CP, i livelli di trombina sono notevolmente aumentati in tessuti infiammati e, in modo particolare, nel fluido sinoviale di pazienti affetti da artrite reumatoide. Difatti, la trombina agisce come mediatore pro-infiammatorio e chemiotattico. In nostri dati indicano che la trombina è in grado di ostacolare, in seguito a proteolisi, l’attività antiossidante della ceruloplasmina. Queste evidenze sperimentali sono state confermate dal fatto che in presenza di irudina il cleavage della CP è inibito e l’infiammazione articolare nei soggetti con artrite reumatoide è ridotta

The Role of Proteolysis in Amyloidosis

Amyloidoses are a group of diseases associated with deposits of amyloid fibrils in different tissues. So far, 36 different types of amyloidosis are known, each due to the misfolding and accumulation of a specific protein. Amyloid deposits can be found in several organs, including the heart, brain, kidneys, and spleen, and can affect single or multiple organs. Generally, amyloid-forming proteins become prone to aggregate due to genetic mutations, acquired environmental factors, excessive concentration, or post-translational modifications. Interestingly, amyloid aggregates are often composed of proteolytic fragments, derived from the degradation of precursor proteins by yet unidentified proteases, which display higher amyloidogenic tendency compared to precursor proteins, thus representing an important mechanism in the onset of amyloid-based diseases. In the present review, we summarize the current knowledge on the proteolytic susceptibility of three of the main human amyloidogenic proteins, i.e., transthyretin, β-amyloid precursor protein, and α-synuclein, in the onset of amyloidosis. We also highlight the role that proteolytic enzymes can play in the crosstalk between intestinal inflammation and amyloid-based diseases