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

Dimerization of human angiogenin and of variants involved in neurodegenerative diseases

Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies

SHBG(141-161) Domain-Peptide Stimulates GPRC6A-Mediated Response in Leydig and \u3b2-Langerhans cell lines

GPRC6A is acknowledged as a major regulator of energy metabolism and male fertility through the action of undercarboxylated osteocalcin (ucOCN), representing a possible therapeutic target. We recently showed that the sex hormone-binding globulin (SHBG) binds to GPRC6A through the likely involvement of the 141-161 domain. To confirm this model, here we investigated the possible binding and agonist activity of SHBG(141-161) domain-peptide (SHBG141-161) on GPRC6A. The binding of SHBG141-161 to GPRC6A and downstream dissociation from G\u3b1i(GDP) protein was computationally modelled. SHBG141-161 was obtained by solid-phase synthesis, characterized by circular dichroism (CD) and the receptor binding was assessed by displacement of ucOCN on HEK-293 cells transfected with GPRC6A gene. Agonist activity of SHBG141-161 was assessed on Leydig MA-10 and Langerhans \uf062-TC6 cell lines through the GPRC6A-mediated release of testosterone (T) and insulin. SHBG141-161 was predicted to bind to GPRC6A and to reduce the affinity for G\u3b1i(GDP) at computational level. Conformational properties and binding to GPRC6A of the synthetic SHBG141-161 were confirmed by CD and displacement experiments. SHBG141-161 stimulated cell secretion of T and insulin, with dose dependency from 10-13 to 10-11M for T release (respectively P=0,041 10-13M; P = 0,032 10-12M; P = 0,008 10-11M vs basal) and for 10-12 to 10-10M for insulin (respectively P=0,041 10-12M; P=0,007 10-11M; P=0,047 10-10M; P=0,045 vs basal). Blockade with anti GPRC6A IgG abolished the response to SHBG141-161, suggesting agonist specificity. SHBG141-161 showed stimulating activity on GPRC6A, representing a template peptide with possible therapeutic use for metabolic and endocrine disorder

A serine protease secreted from Bacillus subtilis cleaves human plasma transthyretin to generate an amyloidogenic fragment

Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1-3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis, a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59-127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart. Peterle et al. show that a subtilisin like serine protease secreted from gut microbiota Bacillus subtilis cleaves the wild-type human transthyretin (hTTR) to generate an amyloidogenic peptide. High propensity of the hTTR fragment to form pathogenic protein aggregates implicates the serine protease in the pathogenesis of acquired senile systemic amyloidosis

Molecular Mechanism in the Alteration of Hemostasis

Hemostasis 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

Molecular Mechanism in the Alteration of Hemostasis

Hemostasis 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

Protein Engineering by Chemical Methods: Incorporation of Nonnatural Amino Acids as a Tool for Studying Protein Folding, Stability, and Function

Proteins are large complex biomolecules that act as the effectors of essentially all cell functions. Due to the intrinsic complexity of protein architecture at the microscopic level and the inadequacy of theoretical methods to predict protein reactivity (ie, folding, stability, and function), protein engineering has emerged as a valuable tool to investigate structure-stability-activity relationships in proteins and nowadays recombinant DNA technologies are the "gold standard" for site-specifically manipulating a given protein chain. The usefulness of current mutagenesis techniques, however, is limited by the relatively poor chemical diversity of the 20 DNA-coded amino acids, such that it is difficult to precisely assign the observed change of protein stability or function to the variation of a single physicochemical property at a protein site (ie, hydrophobicity, conformational propensity, polarizability, hydrogen bonding, etc). In this article, we report relevant examples from our laboratory showing that chemical methods, that is, enzyme-catalyzed semisynthesis and stepwise solid-phase synthesis, allow to conveniently incorporate non-natural amino acids with "tailored" side chains into small proteins and thus effectively transfer the structure-activity relationship methodology, typical of the medicinal chemistry approach on small molecules, to the study of folding, stability, and molecular recognition in macromolecular protein systems

Exogenous human α-Synuclein acts in vitro as a mild platelet antiaggregant inhibiting α-thrombin-induced platelet activation

α-Synuclein (αSyn) is a small disordered protein, highly conserved in vertebrates and involved in the pathogenesis of Parkinson's disease (PD). Indeed, αSyn amyloid aggregates are present in the brain of patients with PD. Although the pathogenic role of αSyn is widely accepted, the physiological function of this protein remains elusive. Beyond the central nervous system, αSyn is expressed in hematopoietic tissue and blood, where platelets are a major cellular host of αSyn. Platelets play a key role in hemostasis and are potently activated by thrombin (αT) through the cleavage of protease-activated receptors. Furthermore, both αT and αSyn could be found in the same spatial environment, i.e. the platelet membrane, as αT binds to and activates platelets that can release αSyn from α-granules and microvesicles. Here, we investigated the possibility that exogenous αSyn could interfere with platelet activation induced by different agonists in vitro. Data obtained from distinct experimental techniques (i.e. multiple electrode aggregometry, rotational thromboelastometry, immunofluorescence microscopy, surface plasmon resonance, and steady-state fluorescence spectroscopy) on whole blood and platelet-rich plasma indicate that exogenous αSyn has mild platelet antiaggregating properties in vitro, acting as a negative regulator of αT-mediated platelet activation by preferentially inhibiting P-selectin expression on platelet surface. We have also shown that both exogenous and endogenous (i.e. cytoplasmic) αSyn preferentially bind to the outer surface of activated platelets. Starting from these findings, a coherent model of the antiplatelet function of αSyn is proposed

Molecular Mapping of \u3b1 -Thrombin( \u3b1 T)/ \u3b2 2-Glycoprotein I(\u3b22GpI) Interaction Reveals How \u3b22GpI Affects \u3b1 T Functions

: \u3b22-Glycoprotein I (\u3b22GpI) is the major autoantigen in the antiphospholipid syndrome, a thrombotic autoimmune disease. Nonetheless, the physiological role of \u3b22GpI is still unclear. In a recent work, we have shown that \u3b22GpI selectively inhibits the procoagulant functions of human a-thrombin (\u3b1T) (i.e. prolongs fibrin clotting time, tc, and inhibits \u3b1T-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 \u3b1T exclusively at the protease catalytic site. Here we used several different biochemical/biophysical techniques and molecular probes for mapping the binding sites in \u3b1T-\u3b22GpI complex. Our results indicate that \u3b1T exploits the highly electropositive exosite-II, which is also responsible for anchoring \u3b1T on the platelet GpIb\u3b1 receptor, for binding to a continuous negative region on \u3b22GpI 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 \u3b22GpI, 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 \u3b1T-\u3b22GpI interaction, which was in agreement with the experimental results. Altogether, these findings help to understand how \u3b22GpI affects \u3b1T interactions and suggest that \u3b22GpI may function as a scavenger of \u3b1T for binding to GpIb\u3b1 receptor, thus impairing platelets aggregation while enabling normal cleavage of fibrinogen and PC