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

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

Burden of caregiving for cardiovascular dysautonomia in Parkinson's disease

PURPOSE: We sought to estimate the impact of cardiovascular autonomic neuropathy (cAN) on informal caregivers of patients with Parkinson’s disease (PD), defined as individuals providing regular care to a friend, partner, or family member with PD, and to evaluate the mutual relationship between caregiver burden and patient health-related quality of life (HRQoL). METHODS: We enrolled 36 consecutive patients with PD and their informal caregivers. Patients underwent a detailed motor, autonomic, cognitive, and functional assessment. Caregivers were assessed using the Zarit Burden Interview (ZBI). Differences in caregiver burden, expressed by the ZBI score, and strength of association between caregiver burden, cAN, and HRQoL were assessed using analysis of covariance (ANCOVA), logistic regression, and linear regression analyses. Analyses were adjusted for patients’ age, PD duration, and motor and cognitive disability, as well as caregivers’ age. RESULTS: Moderate-severe caregiver burden was reported in 41.7% of PDcAN(+) versus 8.7% of PDcAN(−) (p < 0.001). The ZBI score was increased in PDcAN(+) versus PDcAN(−) (31.5 ± 3.4 versus 15.2 ± 2.3; p < 0.001), with tenfold higher odds (p = 0.012) of moderate-severe caregiver burden in PDcAN(+), even after adjusting for potential confounders. The ZBI score correlated with cAN severity (p = 0.005), global autonomic impairment (p = 0.012), and HRQoL impairment (p < 0.001). CONCLUSION: These results highlight the significant impact of cAN on PD caregivers and the need for targeted interventions addressing this frequently overlooked and insufficiently treated source of nonmotor disability in PD

Are patients with GBA-Parkinson disease good candidates for deep brain stimulation? A longitudinal multicentric study on a large Italian cohort

Background: GBA variants increase the risk of developing Parkinson disease (PD) and influence its outcome. Deep brain stimulation (DBS) is a recognised therapeutic option for advanced PD. Data on DBS long-term outcome in GBA carriers are scarce. Objective: To elucidate the impact of GBA variants on long-term DBS outcome in a large Italian cohort. Methods: We retrospectively recruited a multicentric Italian DBS-PD cohort and assessed: (1) GBA prevalence; (2) pre-DBS clinical features; and (3) outcomes of motor, cognitive and other non-motor features up to 5 years post-DBS. Results: We included 365 patients with PD, of whom 73 (20%) carried GBA variants. 5-year follow-up data were available for 173 PD, including 32 mutated subjects. GBA-PD had an earlier onset and were younger at DBS than non-GBA-PD. They also had shorter disease duration, higher occurrence of dyskinesias and orthostatic hypotension symptoms.At post-DBS, both groups showed marked motor improvement, a significant reduction of fluctuations, dyskinesias and impulsive-compulsive disorders (ICD) and low occurrence of most complications. Only cognitive scores worsened significantly faster in GBA-PD after 3 years. Overt dementia was diagnosed in 11% non-GBA-PD and 25% GBA-PD at 5-year follow-up. Conclusions: Evaluation of long-term impact of GBA variants in a large Italian DBS-PD cohort supported the role of DBS surgery as a valid therapeutic strategy in GBA-PD, with long-term benefit on motor performance and ICD. Despite the selective worsening of cognitive scores since 3 years post-DBS, the majority of GBA-PD had not developed dementia at 5-year follow-up