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Molecular Mechanism in the Alteration of Hemostasis
No full textHemostasis is a finely tuned physiological process that, through the concerted action of several blood cells and proteins, maintains the integrity of the vascular system. This stepwise process begins after a vessel wall injury and includes: an initial vasospasm, a platelet plug formation (primary hemostasis), an assembly and activation of the coagulation factors that results in fibrin deposition at the site of injury (secondary hemostasis), and a final dissolution of the fibrin clot that restores the blood vessel patency (fibrinolysis) (Chapter 1). Alterations affecting one or more of these delicate processes lead to a large number of pathological manifestations, commonly referred to as cardiovascular diseases (CVD). Nowadays, CVD are the major cause of mortality and morbidity worldwide. Despite the social and economic burden of CVD, the currently available pharmaceutical repertoire is relatively limited to a few classes of molecules (heparins, platelet antiaggregants, vitamin-K antagonists, direct thrombin inhibitors) which, however, display important side effects and need to be employed with careful dose adjustments. These difficulties stem primarily from: i) the intrinsically complex nature of the procoagulant and anticoagulant biochemical mechanisms leading to physiological hemostasis, which renders external intervention very risky and unpredictable; ii) the inadequate knowledge of the biochemical mechanisms linking blood coagulation to other vital physio-pathological processes.
The general aim of this Ph.D. project was to investigate some of the molecular mechanisms underlying hemostatic disorders. To address this relevant question, we proceeded by studying selected pathologies for which association with hemostatic complications has either been long-established (i.e., Antiphospholipid Syndrome (APS), infectious diseases) or has just been hypothesized (Parkinson’s disease (PD), Transthyretin-related Amyloidosis (ATTR)), focusing our attention on the physio-pathological proteins involved in the onset of these disorders. In a first stage, our attention was focused on the study of novel interactions between α-thrombin (αT), the key enzyme of the coagulation cascade, with other plasma proteins (i.e., β2-glycoprotein-I, α-synuclein). In a second stage, we investigated an alternative mechanism of activation of prothrombin, the precursor of αT, by a bacterial protease (subtilisin from B. subtilis). Finally, some selected proteases were tested against human transthyretin, whose proteolyzed form is a key factor in the onset of ATTR.
In its traditional pathway, blood coagulation culminates with the FXa-mediated conversion of prothrombin zymogen into active αT, through the formation of the prothrombinase complex on the platelet surface. Mature αT is a 36.7 kDa serine protease with a chymotrypsin-like fold. αT plays a pivotal role in blood coagulation, being able to exert both procoagulant (platelets aggregation, fibrin generation) and anticoagulant (protein C activation) functions. The equilibrium between such different activities is regulated by the interaction of αT with other proteins through its active site and two positively charged regions, called exosites (exosite I and exosite II), which flank the catalytic cleft. In addition, αT is a multifunctional protease that, beyond blood coagulation, plays important roles also in other physiological processes such as inflammation, innate immune system, and nervous systems.
In Chapter 2 we mapped the interaction between αT and β2-Glycoprotein I (β2GpI). β2GpI is a heavily glycosylated 45 kDa protein that resides in human plasma at a physiological concentration of 4 µM (0.25 mg/ml). Since the early 90's, β2GpI has been identified as the major autoantigen in the antiphospholipid syndrome (APS), a severe autoimmune disease clinically characterized by hemostatic alterations such as venous and arterial thrombosis, fetal loss and thrombocytopenia. Despite its involvement in the pathogenesis of APS, the physiological roles of β2GpI remain unclear and both pro- and anti-coagulant functions have been reported for this protein. In a recent work, we have shown that β2GpI selectively inhibits the procoagulant functions of human α-thrombin (i.e. prolongs fibrin clotting time, tc, and inhibits α-thrombin-induced platelets aggregation) without affecting the unique anticoagulant activity of the protease (i.e. the proteolytic generation of the anticoagulant protein C). Here, combining molecular modeling with biochemical/biophysical techniques, we provided a coherent structural model of αT-β2GpI complex. The model has allowed us to understand at the molecular level our previous in vitro results. In particular, our findings suggested that β2GpI may function as an anticoagulant protein, acting as a scavenger of αT for the binding to GpIbα receptor, thus impairing platelets aggregation while enabling normal cleavage of fibrinogen and protein C.
Chapter 3 was dedicated to the role of bacterial proteases in inducing blood coagulation by direct proteolytic activation of prothrombin. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies, and, in about 35% of sepsis cases, by disseminated intravascular coagulopathies (DIC). Here, we show that addition of subtilisin (50 nM–2 µM), a serine protease secreted by the nonpathogenic bacterium Bacillus subtilis, to human plasma induces clotting by proteolytically converting prothrombin into active σPre2, a nicked Pre2 derivative with a single cleaved Ala470–Asn471 bond. Notably, we found that this non-canonical cleavage at Ala470–Asn471 is instrumental for the onset of catalytic activity in σPre2, which was however reduced of about 100-200 fold compared with natural αT. Of note, σPre2 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 prothrombin by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis.
The study object presented in Chapter 4 is the interplay between αT and α-synuclein (αSyn). αSyn is a small (14.6 kDa) presynaptic protein mainly synthesized in the brain and whose aggregation has been shown to trigger the onset of different neurodegenerative diseases, commonly referred to as synucleinopathies (i.e., Parkinson disease). As for β2GpI, the exact physiological role of αSyn is still elusive. Intriguingly, αSyn is also synthesized by platelets and was found to inhibit the Ca2+-dependent release of procoagulant α-granules after αT stimulation. Moreover, clinical evidences clearly indicate that patients affected by neurodegenerative disorders have lower risks of ischemic attack. The collateral effects of αSyn in the pathogenesis and its localization on platelet surfaces prompted us to investigate a possible role of it in the hemostatic system. Here, we studied the effects of αSyn on fibrin generation and platelet activation. Furthermore, we mapped the interaction sites on αSyn and αT. Briefly, our results indicate that the negatively charged C-terminal tail of αSyn binds to the electropositive exosite-2 of thrombin, thus impairing αT-mediated platelet activation in whole blood. At variance, αSyn does not alter the rate of fibrin generation, resulting only in a minor change in the ensuing fibrin structure.
In Chapter 5 we attempted to correlate the onset of systemic transthyretin amyloidosis to an altered activation of blood coagulation. Human transthyretin (hTTR) is an abundant homo-tetrameric plasma protein (0.2 mg/ml) involved in the transport of thyroxine and retinol through the binding to retinol binding protein. Beyond its physiological roles, hTTR is known as an amyloidogenic protein whose aggregation is responsible for several amyloid diseases, including senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), and familial amyloid cardiomyopathy (FAC). From a mechanistic point of view, the proteolytic cleavage of hTTR represents an important step in fibril formation. In particular, after cleavage around position 50, hTTR C-terminal fragments have been found to aggregate far more efficiently than the full-length hTTR. Nowadays, the protease(s) responsible for this cleavage is yet to be identified although it is predicted to be a serine protease with a trypsin-like fold. Since all coagulation factors are trypsin-like serine proteases, we decided to probe them for the proteolytic cleavage of hTTR. In addition, we also probed some selected bacterial proteases, as well as some digestive apparatus and immune system proteases. hTTR was resistant to all proteases tested except to subtilisin from B. subtilis, which was able to cleave hTTR at pH 7.4, generating in high yields the amyloidogenic fragment hTTR(59-127). Since the hTTR(59-127) fragment was identified in amyloid deposits, these new insights might have relevant implications in hTTR-based amyloidosis
Noncoded 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
Molecular Mapping of α -Thrombin( α T)/ β 2-Glycoprotein I(β2GpI) Interaction Reveals How β2GpI Affects α T Functions
No full text: β2-Glycoprotein I (β2GpI) is the major autoantigen in the antiphospholipid syndrome, a thrombotic autoimmune disease. Nonetheless, the physiological role of β2GpI is still unclear. In a recent work, we have shown that β2GpI selectively inhibits the procoagulant functions of human a-thrombin (αT) (i.e. prolongs fibrin clotting time, tc, and inhibits αT-induced platelets aggregation) without affecting the unique anticoagulant activity of the protease, i.e. the proteolytic generation of the anticoagulant protein C (PC) from the PC zymogen, which interacts with αT exclusively at the protease catalytic site. Here we used several different biochemical/biophysical techniques and molecular probes for mapping the binding sites in αT-β2GpI complex. Our results indicate that αT exploits the highly electropositive exosite-II, which is also responsible for anchoring αT on the platelet GpIbα receptor, for binding to a continuous negative region on β2GpI structure, spanning domain IV and (part of) domain V, while the protease active site and exosite-I (i.e. the fibrinogen binding site) remain accessible for substrate/ligand binding. Furthermore, we provided evidence that the apparent increase in tc, previously observed with β2GpI, is more likely caused by alteration of the ensuing fibrin structure rather than by inhibition of fibrinogen hydrolysis. Finally, we produced a theoretical docking model of αT-β2GpI interaction, which was in agreement with the experimental results. Altogether, these findings help to understand how β2GpI affects αT interactions and suggest that β2GpI may function as a scavenger of αT for binding to GpIbα receptor, thus impairing platelets aggregation while enabling normal cleavage of fibrinogen and PC
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Non-canonical proteolytic activation of human prothrombin by subtilisin from Bacillus subtilis may shift the procoagulantâ anticoagulant equilibrium toward thrombosis
Full text linkBlood 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 –Ile-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 –Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470 –Asn-471 is instrumental for the onset of catalysis in Pre2, which was, however, reduced about 100 –200-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–anticoagulant equilibrium toward thrombosis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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