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Background Rivaroxaban (Riva), a direct FXa inhibitor, at 2.5 mg twice-daily (bid) combined with low-dose aspirin (ASA, 100 mg once daily-od) reduced major vascular events vs. ASA alone in subjects with stable coronary artery (CAD) or symptomatic peripheral artery disease (PAD). Whether this benefit is due to the anticoagulant effect or additional FXa-mediated effects through the platelet and endothelial cell thrombin receptors is unknown. Type 2 diabetes mellitus (T2DM) is characterized by high platelet activation and oxidative stress that may contribute to increased cardiovascular risk. Purpose We investigated the effects of Riva (2.5 mg bid) + ASA (100 mg od) vs. ASA (100 mg od) alone on platelet and oxidative biomarkers in subjects with T2DM and stable vascular disease (stable CAD, symptomatic PAD and/or significant carotid stenosis). Methods In this randomized, open-label, cross-over trial, patients were randomized to continue ASA for 4 weeks and then add Riva for 4 weeks, or add Riva in the first 4 weeks and then continue with ASA alone for 4 weeks. Primary endpoints were: in vivo platelet activation and lipid peroxidation, assessed by the urinary excretion of 11-dehydro-TXB2 (TXM) and 8-iso-PGF2alpha (ISOP), respectively. Secondary endpoints included: routine coagulation tests, D-dimer, thrombin generation, serum TXB2, and Riva plasma concentrations. Results Seventy-6 subjects (10 females) were recruited: age 68±7 years (mean±SD); BMI 27.1±3.5 kg/m2; fasting glucose 129±31 mg/dL; HbA1c 6.8±0.9%; serum creatinine 1±0.25 mg/dL; LDL-cholesterol 77±31 mg/dL. Two patients dropped out: one for benign, self-limiting hematuria, one for unwillingness to continue, 8 subjects are completing the study leaving 66 who completed the 8-week randomized treatment and showed no sequence effect. Urinary TXM and ISOP were significantly reduced by Riva+ASA vs. ASA alone: TXM was 260 [195–398] vs. 335 [225–441] pg/mg creatinine and ISOP 722 [601–991] vs. 827 [648–1350] pg/mg creatinine (median [IQR]) on Riva+ASA vs. ASA alone, respectively (p Conclusions In ASA-treated subjects with T2DM and stable vascular disease, the addition of very low-dose Riva restrained incompletely-suppressed lipid peroxidation and platelet activation and modified the kinetics of thrombin formation. These changes may contribute to the beneficial effects of the Riva+ASA combination. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Investigator-initiated study funded by Bayer AG

We concisely review clinical, autopsy, experimental and molecular data of 2019 coronavirus disease (COVID-19). Angiotensin-converting enzyme 2 disruption and thromboinflammatory microangiopathy emerge as distinctive features. Briefly, entry of the virus into microvessels can profoundly disrupt the local renin-angiotensin system, cause endothelial injury, activate the complement cascade and induce powerful thromboinflammatory reactions, involving, in particular, von Willebrand factor, that, if widespread, may lead to microvascular plugging, ischemia and, ultimately, organ failure. We believe the current COVID-19 data consolidate a widely unrecognised paradigm of potentially fatal thromboinflammatory microvascular disease., Highlights • SARS-CoV-2 entry into microvessels can disrupt the local renin-angiotensin-system. • Complement and von Willebrand factor overexpression occur in COVID-19. • COVID-19 patients show widespread thromboinflammatory microvascular disease. • COVID-19 provides an important paradigm of potentially fatal microangiopathy.

On-pump cardiac surgery may trigger inflammation and accelerate platelet cyclooxygenase-1 renewal, thereby modifying low-dose aspirin pharmacodynamics. Thirty-seven patients on standard aspirin 100 mg once-daily were studied before surgery and randomized within 36 hours postsurgery to 100 mg once-daily, 100 mg twice-daily, or 200 mg once-daily for 90 days. On day 7 postsurgery, immature and mature platelets, platelet mass, thrombopoietin, glycocalicin, leukocytes, C-reactive protein, and interleukin-6 significantly increased. Interleukin-6 significantly correlated with immature platelets. At day 7, patients randomized to 100 mg once-daily showed a significant increase in serum thromboxane (TX)B2 within the 24-hour dosing interval and urinary TXA2 metabolite (TXM) excretion. Aspirin 100 mg twice-daily lowered serum TXB2 and prevented postsurgery TXM increase (P < 0.01), without affecting prostacyclin metabolite excretion. After cardiac surgery, shortening the dosing interval, but not doubling the once-daily dose, rescues the impaired antiplatelet effect of low-dose aspirin and prevents platelet activation associated with acute inflammation and enhanced platelet turnover.

Background Polypharmacy in the elderly is a matter of concern, especially regarding adverse events and drug–drug interactions. Little is known about real world drug use in the elderly in Italy. The present study aimed at describing drug use in the elderly with respect to individual characteristics, performing a cross-sectional population study in Lazio, Italy. Methods From the regional health care assistance file, residents aged 65 years and older (mean age = 75.9 years, SD = 7.4) and alive at the end of 2010 were enrolled. Drug consumption was linked from the drug claims registry for 2010, and hospital admissions were retrieved from the hospital admission registry during 2009–2010. Enrolees were characterised for socio-demographic variables, presence of chronic diseases, and drug consumption, considering large groups, and specific sub-groups. Polypharmacy was defined as use of 6+ drugs (ATC 4th level). Results Of the 1,122,864 elderly residents, 57.9% were women, and 48.8% aged 75+. In the previous two years, 28.3% had been admitted to hospital; most common causes were cerebro- and cardiovascular diseases, diabetes and tumours; 78.9% of the elderly population of the city of Rome lived in low-level socio-economic areas. Only 6.8% had not been prescribed drugs in the 12-months observation period, and 58.8% were exposed to polypharmacy. With respect to the overall population, polypharmacy users were more often females (59.7%), older (54.5% aged 75 + ), had poorer health conditions (37.2% hospitalised), and more often lived in poorer areas (19.9%). Conclusion In Lazio, polypharmacy is an important phenomenon in the elderly population. Female gender, older ages and presence of chronic diseases are related to multiple drug use. The present results offer a useful tool for future initiatives aiming at reducing inappropriate polypharmacy and target interventions to subpopulations at higher risk of being exposed to polypharmacy.

Background At early stages, SSc lung involvement is characterised by Ground Glass Opacities (GGO) at High Resolution Computed Tomography (HRCT). However, HRCT scan is not able to provide functional information and to discriminate between an “active inflammatory” and an “established fibrotic” GGO. 18Fluoro-Deoxy-d-Glucose (18F-FDG) Positron Emission Tomography/Computed Tomography (PET/CT) is able to detect metabolic activity picking up inflammation and provides both morphologic and metabolic data. Objectives The aim of this study was to evaluate, if 18F-FDG PET/CT scan may identify the inflammatory component of GGO in SSc interstitial lung disease. Methods Seven patients with SSc (1 male and 6 females; mean age 59.56 y±9.15 SD; median disease duration 5 years, 2–11 who underwent 18F-FDG PET/CT scan because of cancer screening, were retrospectively analysed. HRCT images analysis led to classification of pulmonary segments as “negative” (normal morphology) and ”positive” (GGO). Furthermore, the “Warrick score” was used as a staging tool for SSc-ILD. Mean Standardised Uptake Value (mSUV) of segmental parechima was normalised (nmSUV) by comparison with the values of selected control subjets. Results No SSc patient was affected by cancer. Three patients had a Warrick Score >0, while 4 patients did not had any lung involvement (Warrick Score=0). The 3 patients with a Warrick Score >0 had also skin involvement with a median mRSS 6 (2–7) and pathological lung FDG uptake. In “positive” segments of SSc patients, nmSUV was significantly higher than in the lung segments of the control population (mean estimation 1.53; C.I. 1.42–1.65, p 0, the nmSUV was significantly higher than in segments of the control population (mean estimation 1.29; C.I. 1.22–1.37, p 0. “Negative” lung segments of patients with Warrick Score >0 showed a 32% higher 18F-FDG uptake than “negative” lung segments of patients with Warrick Score=0. (C.I. 0.17–0.48, p Conclusions Morphologically “positive” GGO segments show an increased 18F-FDG uptake suggesting the existence of a metabolically active (inflammatory) GGO. However, in patients with GGO, negative lung segments showed a higher nmSUV than negative lung segments in patients without GGO. This may suggest that PET/CT may disclose an underlying inflammatory process, which cannot yet be evidenced by HRCT. Further studies on a larger population are warranted to confirm these data and possibly provide a prognostic significance of PET/CT positivity in SSc patients. Disclosure of Interest None declared

Summary Background Essential thrombocythemia (ET) is characterized by increased platelets and prevalent thrombosis. An acquired von Willebrand factor (VWF) disease has been hypothesized and inconsistently associated with extreme thrombocytosis or rare bleeding in ET. Whether VWF is modified in ET patients with controlled platelet count remains unclear. Objectives We studied different VWF- and platelet-associated parameters in ET patients treated according to current recommendations. Patients/Methods Sixty-nine ET patients (M = 29; median age, 62 [48–70] years; platelets, 432 [337–620] × 103 μL−1), 69 matched controls and 10 subjects with reactive thrombocytosis (RT) were studied. VWF:antigen (Ag), activity (act), electrophoretic patterns, VWF:propeptide, plasma glycocalycin (GC), glycoproteinV (GpV), ADAMTS-13, elastase, C-reactive protein and serum thromboxane (TX)B2 were measured. Results In ET patients, VWF:Ag was increased by 31 ± 13% vs. controls (P

Summary Background This work was aimed at characterizing the interaction of β2-glycoprotein I (β2GPI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagulation cascade. Methods The β2GPI–thrombin interaction was studied by surface plasmon resonance (SPR), fluorescence, and molecular modeling. The effect of β2GPI on the procoagulant (fibrin generation and platelet aggregation) and anticoagulant (protein C activation) functions of thrombin were investigated with turbidimetric, immunocytofluorimetric and enzymatic assays. Results SPR and fluorescence data indicated that β2GPI tightly bound thrombin (Kd = 34 nm) by interacting with both protease exosites, while leaving the active site accessible. This picture is fully consistent with the theoretical model of the β2GPI–thrombin complex. In particular, blockage of thrombin exosites with binders specific for exosite-1 (hirugen and HD1 aptamer) or exosite-2 (fibrinogen γ′-peptide and HD22 aptamer) impaired the β2GPI–thrombin interaction. Identical results were obtained with thrombin mutants having one of the two exosites selectively compromised by mutation (Arg73Ala and Arg101Ala). Fluorescence measurements indicated that β2GPI did not affect the affinity of the enzyme for active site inhibitors, such as p-aminobenzamidine and the hirudin(1–47) domain, in agreement with the structural model. β2GPI dose-dependently prolonged the thrombin clotting time and ecarin clotting time in β2GPI-deficient plasma. β2GPI inhibited thrombin-induced platelet aggregation (IC50 = 0.36 μm) by impairing thrombin cleavage of protease-activated receptor 1 (PAR1) (IC50 = 0.32 μm), both on gel-filtered platelets and in whole blood. Strikingly, β2GPI did not affect thrombin-mediated generation of the anticoagulant protein C. Conclusions β2GPI functions as a physiologic anticoagulant by inhibiting the key procoagulant activities of thrombin without affecting its unique anticoagulant function.

Background: Clinically apparent Alzheimer's disease (AD) is thought to result when brain tissue damage exceeds a critical threshold of "brain reserve", a process possibly accelerated by the apolipoprotein E (ApoE) E4 allele. The interaction between onset age and ApoE genotype was investigated to assess whether early disease onset (

Declines in brain glucose metabolism have been described early in Alzheimer's disease (AD), and there is evidence that a genetic predisposition to AD contributes to accelerate this process. The epsilon 4 (e4) allele of the apolipoprotein E (ApoE) gene has been implicated as a major risk factor in this process. The aim of this FDG-PET study was to assess the ApoE e4 dose related effect on regional cerebral glucose metabolism (METglc) in clinical AD patients, with statistical voxel based methods.Eighty six consecutive mild to moderate AD patients included in the Network for Efficiency and Standardisation of Dementia Diagnosis database underwent FDG-PET scans at rest. PCR was used to determine the ApoE genotype. Patients were grouped as e4 non-carriers (n = 46), e3/e4 (n = 27) and e4/e4 (n = 13) carriers. A voxel-based mapping program was used to compare each AD subgroup with a database of 35 sex and age matched controls (p0.001, corrected for cluster extent) and also to compare between the subgroups (p0.001, uncorrected).No difference was found as to age at examination, age at onset, sex, disease duration, educational level, or severity of dementia between AD subgroups. Compared with controls, all AD subgroups had equivalent METglc reductions in the precuneus, posterior cingulate, parietotemporal, and frontal regions. Direct comparisons between AD subgroups indicated that patients with at least one e4 allele had METglc reductions within additional associative and limbic areas compared with e4 non-carriers.The present FDG-PET study showed different metabolic phenotypes related to the ApoE genotype in clinical AD patients, as revealed with voxel based statistical methods. The results suggest a generalised disorder in e4 carriers impairing metabolism globally, in addition to the more localised changes typical of AD patients.

The activation of human platelets by alpha-thrombin is mediated in part by cleavage of the protease-activated receptor (PAR) 1 and 4 and by the glycoprotein (Ib)alpha, (Gp(Ib)alpha), which binds with high affinity to alpha-thrombin. Recent studies have shown that the thrombin domain referred to as heparin binding site (HBS) is involved in the interaction with the platelet Gp(Ib)alpha. The HBS is rich in basic amino acids. To identify the key amino acid residues involved in the binding to Gp(Ib)alpha, we have performed alanine scanning mutagenesis of the basic HBS R93, R97, R101, R233, K236, K240, R233/K236/Q239, as well as of the neutral Q239 residues, located in different regions of the domain. For comparison, mutation at R67 within the fibrinogen recognition site (FRS) of thrombin was performed as well. Solid-phase binding experiments showed that the Kd of thrombin-GpIb interaction was reduced 22-fold for R93A, 8-fold for R97A, 13-fold for R101A, 29-fold for R233A, 21-fold for K236A, 5-fold for K240A, and 31-fold for the triple mutant R233A/K236A/Q239A, while the Q239A and R67A forms did not show any significant affinity change. The platelet activating capacity of these mutants was evaluated as well. Using gel-filtered platelets, the EC50 value of thrombin-induced aggregation was from 5- to 13-fold higher in the HBS mutants than in the WT form, and was linearly and positively correlated with the corresponding Kd values pertaining to thrombin binding to GpIb. Measurements of PAR-1 hydrolysis on the platelet membrane showed that the HBS mutants R233A, R101A, R93A, K236A, and R233/K236/Q239 forms had a reduction of the apparent kcat/Km value. These results are a consequence of a defective binding to GpIb, which is known to optimize the interaction with PAR-1 in situ. A confirm of this hypothesis came from the demonstration that the kcat/Km value pertaining to the hydrolysis by the HBS-mutated thrombins of the synthetic PAR-1 38-60 peptide in solution was similar to that one obtained with the WT form. In conclusion, these experiments provide a structural and functional mapping of the thrombin HBS subregions involved in the binding to the platelet Gp(Ib)alpha and in the cell activation.

SummaryThe myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This study investigated the biochemical and functional effects of α-thrombin oxidation by MPOS. This system, in the presence of 100 µM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCl, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human α-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin. Abbreviations: DNPH: 2,4-dinitrophenylhydrazine; FpA: fibrinopeptide A; FpB: fibrinopeptide B; FRS: Fibrinogen recognition Site; GpIb: Glycoprotein Ib; HBS: Heparin Binding Site; MPO: myeloperoxidase; MPOS: Myeloperoxidase System; Pip: Pipecolynic acid; PMN: polymorphonuclear cells; pNA: para-nitroaniline; PPACK: Phenylalanine-Proline-Arginine-Chloromethyl-Ketone; TM: Thrombomodulin; TNB: 5-thio-2-nitrobenzoic acid; TPCK: N-tosyl-L-Phenylalanine-Chloromethyl-Ketone. Thrombin residues are numbered according to the chymotrypsin numbering system (31).

Sirs: Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inclusion disorder with an estimated prevalence among men > 50 years of age up to 1 in 3000 in the general population [16]. The neurologic phenotype of FXTAS is variable and includes intention tremor, ataxia, parkinsonism, and cognitive decline [7–10]. We report evidence from MRI and SPECT that the fragile X premutation determines a subcortical gray matter degeneration involving the midbrain and striatum associated with parkinsonian signs. We studied five men with FXTAS and documentation of the premutation by DNA testing whose genetic and clinical data are detailed in Table 1. In all patients prior MRI showed characteristic symmetric areas of signal changes in the peridentate, middle cerebellar peduncle and cerebral white matter and diffuse brain atrophy [2, 5]. MRI was obtained in patients 1, 2, 4 and 5 at 1.5 T with a protocol including 1 mm thick T1-weighted 3D gradient echo images and 5 mm thick T2-weighted spin-echo (SE) images for evaluation of hypointensities suggestive for possible iron deposits in the basal ganglia [6, 14]. Bulk and shape of the midbrain were evaluated subjectively [12] and midbrain anteroposterior diameter was measured in patients and in 12 healthy subjects [15]. Basal ganglia signals in T2-weighted SE images were visually assessed. Midbrain evaluation in case 4 was performed on hard copies of prior MRI. SPECT examinations were obtained in patients 1, 3–5. They were injected with 111–185 MBq of [123I]FP-CIT for evaluation of striatal dopamine transporter (DAT) density. Patient 3 was also injected with 160 MBq of [123I] idobenzamide (IBZM) for evaluation of striatal dopamine D2 receptors. SPECT examinations were evaluated visually [1]. MRI and SPECT findings are reported in Table 1. Atrophy of the midbrain with an AP diameter smaller than 17 mm [15] was observed in all patients. The superior profile of the midbrain was concave in two patients (Fig. 1) and linear in three. Symmetric hypointensity in the putamen and caudate in T2-weighted SE images was present in all four patients examined with the protocol. DATscan SPECT in patients 3, 4 and 5 showed an asymmetric decrease of the striatum tracer uptake which was mild in cases 4 and 5 and marked in case 3 (Fig. 1). IBZM SPECT in patient 3 revealed almost complete absence of striatal tracer uptake (Fig. 1) Parkinsonism is one of the most common other diagnoses given to FXTAS patients which also include tremor, ataxia, dementia or stroke [9] and fragile X premutation is found in 4 % of patients with diagnosis of probable multi-system atrophy cerebellar type (MSA-C) [10]. Neuropathological examination in FXTAS demonstrates, along with spongiosis of the white matter, nuclear eosinophilic inclusions in the neurons and astrocytes of the cortical and subcortical gray matter, including the substantia nigra and putamen, of possible pathogenetic role [7]. We found MRI evidence of midbrain and striatal neurodegeneration in five patients with FXTAS who showed extra-pyramidal signs. Three patients had vertical gaze palsy, namely a feature of midbrain involvement which is not observed in Parkinson disease (PD) and is characteristic of progressive supranuclear palsy (PSP). In particular, we measured atrophy of the midbrain in all five FXTAS patients that was accompanied by a concave or linear superior profile of the midbrain, instead of the convex profile observed in healthy subjects [12]. These features were emphasized as distinctive of PSP [12, 15]. It is noteworthy that also in LETTER TO THE EDITORS

Shear stress triggers conformational stretching of von Willebrand factor (VWF), which is responsible for its self-association and binding to the platelet receptor glycoprotein (GP)Ibα. This phenomenon supports primary hemostasis under flow. Type 2B VWF natural mutants are considered to have increased affinity for platelet GPIbα.To assess the mechanism responsible for the enhanced interaction of the p.R1306W VWF mutant with the platelet receptor.The interaction of GPIbα with wild-type (WT) and p.R1306W VWF multimers and A1-A2-A3 constructs was investigated with surface plasmon resonance spectroscopy. Analysis of the static VWF conformation in solution was performed with dynamic light scattering spectroscopy. The shear stress-induced self-association of VWF multimers was investigated with atomic force microscopy (AFM) over a 0-60 dyn cm(-2) range.WT VWF did not interact with GPIbα under static conditions, whereas the mutant at ~ 2 μg mL(-1) already bound to the receptor. By contrast, the WT and p.R1306W-A1-A2-A3 constructs showed comparable affinities for GPIbα (Kd ~ 20 nm). The hydrodynamic diameter of resting R1306W VWF multimers was significantly greater than that of the wild type (210 ± 60 nm vs. 87 ± 22 nm). At shear forces of 14 dyn cm(-2) , the p.R1306W multimers rapidly changed conformation, entering a regime of self-aggregation, which, in contrast, was induced for WT VWF by shear forces of 30 dyn cm(-2) . Mechanical stretching AFM experiments showed that p.R1306W multimers needed less energy per length unit (~ 10 pN) to be stretched than the WT protein.The increased affinity of p.R1306W VWF for GPIbα arises mostly from higher sensitivity to shear stress, which facilitates exposure of GPIbα binding sites.

Summary. Background: Binding of von Willebrand factor (VWF) multimers of ultra-large size to platelets is considered the triggering mechanism of microvascular thrombosis in thrombotic thrombocytopenic purpura (TTP). Objective: To assess the potential of VWF-related measurements as markers of disease activity and severity in TTP. Methods: VWF antigen (VWF:Ag), platelet glycoprotein-Ib-α binding-conformation (GPIb-α/BC) and multimeric pattern were investigated in 74 patients with acquired TTP during acute disease, remission or both and 73 healthy controls. In patients with both acute and remission samples available, VWF ristocetin co-factor activity (VWF:RCo) and collagen binding (VWF:CB) were also measured. The relationships of study measurements with the presence of acute disease and remission and with markers of disease severity were assessed. Results: VWF:Ag and VWF-GPIb-α/BC were higher in TTP patients than controls (P

The enzyme indoleamine 2,3-dioxygenase (IDO, EC 1.13.11.42) belongs to the family of heme-containing oxidoreductases and catalyzes the first and rate-limiting step in the kynurenine pathway, the major pathway of tryptophan metabolism. IDO is folded into one large and one small distinct α-helical domains, with the heme prosthetic ring positioned between them. The enzyme, through the oxidative properties of the Fe(3+) atom present at the centre of the heme ring, catalyses the oxidative cleavage of the pyrrole ring of L-Trp to generate N-formyl-kynurenine. The active IDO conformer exists only in the presence of reducing cofactors (such as cytochrome b(5)), requiring the single electron reduction of ferric-to-ferrous iron (Fe(3+)→Fe(2+)), which facilitates binding of L-Trp and O(2) to the enzyme active site. IDO, through production of kynurenine and other downstream metabolites, can regulate immune responses, suppressing effector T-cell function and favouring the differentiation of regulatory T cells. Local expression of the enzyme during inflammation is another self-protection mechanism, which limits antigen-specific immune responses, especially in some organs, as the central nervous system. The detailed knowledge of the structural and functional properties of IDO, was a fundamental step to design and develop new molecules for the pharmacological inhibition of IDO activity in several clinical settings.

Aims/hypothesis High mobility group box 1 (HMGB1) is a cytokine with a key role in tissue regeneration and angiogenesis. Previous studies have shown that topical application of HMGB1 to skin wounds of mouse models of diabetes enhanced vessel density and accelerated wound healing, suggesting that diabetes may affect endogenous HMGB1 functions. Dipeptidyl peptidase IV (DPP-IV/CD26) is a protease whose activity is increased in diabetes and whose inhibition improves glucose tolerance. Since HMGB1 contains potential DPP-IV cleavage sites, we determined whether HMGB1 may be a substrate for DPP-IV and whether DPP-IV-mediated cleavage may alter the biological activity of HMGB1.

A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues 54-65) [hir-(54-65)] domain on the binding and hydrolysis of the three substrates. The results showed that the C-terminal hir-(54-65) fragment affects only the acylation rate, which is increased approx. 1.2-fold for all the substrates. Analysis of the dependence of acylation rate constants on hirudin-fragment concentration, allowed the determination of the equilibrium binding constant of C-terminal hir-(54-65) (Kd approximately 0.7 microM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a Ki which is in excellent agreement with the equilibrium constant derived from viscosity experiments. These results demonstrate that binding of hir-(54-65) to the fibrinogen recognition site of thrombin does not affect the equilibrium binding of amide substrates, but induces only a small increase in the acylation rate of the hydrolysis reaction.

The amidase activity of human gamma-thrombin has been studied in the pH range 6-10 as a function of NaCl concentration and temperature. As recently found for human alpha-thrombin [Di Cera, E., De Cristofaro, R., Albright, D.J., & Fenton, J.W., II (1991) Biochemistry 30, 7913-7924], the Michaelis-Menten constant, Km, shows a bell-shaped dependence over this pH range with a minimum around pH 7.9 in the presence of 0.1 M NaCl at 25 degrees C. The catalytic constant, kcat, has a bell-shaped pH dependence with a maximum around pH 8.6. A thermodynamic analysis of these parameters has enabled a characterization of the linkage between proton and substrate binding, its dependence on NaCl concentration, and the relevant entropic and enthalpic contributions to binding and catalytic events. Three groups seem to be responsible for the control of gamma-thrombin amidase activity as a function of pH. One of these groups has pK values that are significantly different from those found for alpha-thrombin, and all groups show slightly perturbed enthalpies of ionization. The dependence of gamma-thrombin amidase activity on NaCl concentration is different from that of alpha-thrombin. Increasing NaCl concentration always decreases the substrate affinity for the enzyme in the case of alpha-thrombin, regardless of pH. In the case of gamma-thrombin, such an effect is observed only in the pH range 7.5-9, and a reversed linkage is observed at pH less than 7 and greater than 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Steady-state measurements of synthetic substrate hydrolysis by human alpha-thrombin in the presence of human fibrinogen, under experimental conditions where light scattering due to the formation of fibrin aggregates is negligible, have allowed for a quantitative evaluation of Km for fibrinogen. Measurements of Km for fibrinogen carried out at pH 7.5 and 37 degrees C as a function of NaCl, NaBr, KCl, and KBr concentration, from 50 to 500 mM, show that the derivative d ln Km/d ln a +/-, where a +/- is the mean ion activity, is constant over the entire range of salt concentrations and is strictly dependent on the particular salt present in solution. The values of d ln Km/d ln a +/- are found to be equal to 0.75 +/- 0.03 (NaCl), 0.90 +/- 0.01 (NaBr), 0.62 +/- 0.07 (KCl), and 0.60 +/- 0.03 (KBr). Measurements of Km for two synthetic amide substrates, under identical solution conditions, reveal practically no change in Km with salt concentration, while they show a significant decrease in kcat when Na+ salts are replaced by K+ salts. The drastic difference in the salt dependence of Km between fibrinogen and the synthetic amide substrate points out that a significant role may be played by the fibrinogen recognition site in the energetics of thrombin-fibrinogen interaction. The sensitivity of Km for fibrinogen to different salts unequivocally demonstrates that specific ion effects, rather than nonspecific ionic strength effects, modulate thrombin-fibrinogen interaction under experimental conditions of physiological relevance. Analysis of ion effects on clotting curves obtained at pH 7.5 and 37 degrees C also shows a drastic differential effect of cations and anions.(ABSTRACT TRUNCATED AT 250 WORDS)

The enzyme indoleamine 2,3-dioxygenase (IDO) regulates immune responses through the capacity to degrade the essential amino acid tryptophan into kynurenine and other downstream metabolites that suppress effector T-cell function and favour the differentiation of regulatory T cells. Considerable experimental evidence indicates that IDO can be expressed by dendritic cells, by tumour cells or by surrounding stromal cells, either within proximity of the tumour or at distal sites. Recent advances in the biochemistry of IDO and in our understanding of the biological relevance of IDO-mediated tryptophan consumption to the establishment of dominant immune tolerance to cancer will be summarised and discussed. Within the wider context of cancer immunotherapy, this Review also delineates how IDO could be exploited as a molecular target for therapeutic intervention in order to boost anti-cancer immunity.

[18F]-2D-fluoro-deoxiglucose positron emission tomography (FDG-PET) investigates the cerebral metabolic rate of glucose (CMRglc), which is affected by aging and Alzheimer’s disease (AD), showing a specific distribution pattern. FDG-PET is currently the most promising imaging technique for an early diagnosis of AD. This article describes the basic pathophysiological and clinical data needed to understand FDG-PET in AD and to differentiate AD from healthy aging. Methods of interpreting FDG-PET images in AD are described as well. CMRglc changes in relation to genetic factors of AD are also reported.

In summary, hydatidosis is a common infection that infrequently involves the skeleton. When this happens the prognosis is uncertain because of the possible extension of the disease and serious complications, (e.g., pathological fractures, visceral involvement, neurologic damage from vertebral involvement). For this reason, simple curettage is not sufficient for cure and no medical treatment can surely eradicate the disease. Hydatidosis, in our opinion, should be staged and surgically treated like a malignant tumor (e.g., with wide surgical margins). Medical treatment is useful in association with surgery and remains the only therapy for inoperable lesions. A case of echinococcosis of the hip joint in a 37-year-old man has been reported. In this case the clinicoradiographical features suggested pigmented villonodular synovitis. Proof was obtained on the basis of a frozen section on biopsy. The clinical and laboratory characteristics of hydatidosis were discussed, with emphasis on the medical and surgical treatment of the disorder.

Summary. Background: We report a novel case of gray platelet syndrome (GPS). A 14-year-old boy had bleeding diathesis, mild thrombocytopenia, giant platelets with severe defect of α-granule secretory proteins, myelofibrosis and splenomegaly. Methods and results: Platelet function studies showed a marked reduction of aggregation and Ca2+ mobilization by thrombin, protease-activated receptor 1 (PAR1)-activating peptide (AP) and PAR4-AP, PAR1 expression at 55% of normal levels, and a more than two hundred fold reduction of in vitro whole-blood thromboxane B2 (TXB2) production. Sequencing of coding regions of the PAR1 gene failed to show abnormalities. This patient was initially classified as a sporadic case of GPS, as electron microscopy failed to identify giant platelets and/or α-granule deficiency in his relatives. However, further studies on the father and three other relatives showed a relative lack of platelet α-granule proteins by immunofluorescence microscopy, a defective platelet response to PAR4-AP, and severely reduced in vitro whole-blood TXB2 production. On this basis, we suggest that in this family, GPS was transmitted in a dominant fashion with highly variable penetrance. Conclusions: Our study suggests that current diagnostic criteria fail to identify some patients with a mild GPS phenotype and that such patients might be identified by the methods cited above. It also better characterizes the pathogenesis of defective platelet responses to thrombin, and raises interesting questions on the correlation between abnormal PAR function and the lack of α-granule content in GPS.

Objectives: To investigate whether the combination of fluoro-2-deoxy-d-glucose (FDG) PET measures with the APOE genotype would improve prediction of the conversion from mild cognitive impairment (MCI) to Alzheimer disease (AD).Method: After 1 year, 8 of 37 patients with MCI converted to AD (22%). Differences in baseline regional glucose metabolic rate (rCMRglc) across groups were assessed on a voxel-based basis using a two-factor analysis of variance with outcome (converters [n = 8] vs nonconverters [n = 29]) and APOE genotype (E4 carriers [E4+] [n = 16] vs noncarriers [E4−] [n = 21]) as grouping factors. Results were considered significant at p < 0.05, corrected for multiple comparisons.Results: All converters showed reduced rCMRglc in the inferior parietal cortex (IPC) as compared with the nonconverters. Hypometabolism in AD-typical regions, that is, temporoparietal and posterior cingulate cortex, was found for the E4+ as compared with the E4− patients, with the E4+/converters (n = 5) having additional rCMRglc reductions within frontal areas, such as the anterior cingulate (ACC) and inferior frontal (IFC) cortex. For the whole MCI sample, IPC rCMRglc predicted conversion to AD with 84% overall diagnostic accuracy (p = 0.003). Moreover, ACC and IFC rCMRglc improved prediction for the E4+ group, yielding 100% sensitivity, 90% specificity, and 94% accuracy (p < 0.0005), thus leading to an excellent discrimination.Conclusion: Fluoro-2-deoxy-d-glucose-PET measures may improve prediction of the conversion to Alzheimer disease, especially in combination with the APOE genotype.

Alzheimer's disease (AD) is a brain disorder characterized by reduced cerebral glucose metabolism (CMRgl) in several cortical regions. Evidence from neuropathology studies, animal models of AD, and (18)F-FDG PET studies on cognitive impairment suggest that disrupted connections with the entorhinal cortex (EC) could be implicated in the emergence of the cortical hypometabolism. This (18)F-FDG PET study assessed the functional interactions-that is, the intercorrelations between the EC and the whole brain in vivo-in normal aging and AD.Eighty-seven consecutive clinical AD patients underwent (18)F-FDG PET scanning at rest. Thirty-five sex- and age-matched healthy elderly subjects were studied as controls (NC). A voxel-based correlation analysis was performed with statistical parametric mapping to assess significant correlations between relative CMRgl (rCMRgl) in the EC and the rest of the brain, for NC and AD patients. Results were considered significant at P0.001.The pattern of EC functional interactions varies between normal aging and AD patients. In NC, the left and right EC were bilaterally correlated with several cortical and limbic regions, in accord with the major anatomic pathways identified in nonhuman primates. Alternatively, in AD patients, the EC correlations with the contralateral hemisphere were entirely lost, whereas those within the ipsilateral hemisphere were preserved only with the inferior temporooccipital (T-O) areas.This (18)F-FDG PET correlation study indicates that AD-related processes lead to an altered functional relationship between the EC and several cortical and limbic regions, with respect to normal aging. Our results suggest that the assessment of coupled rCMRgl reductions between the EC and the ipsilateral T-O cortex, besides the typical pattern of cortical reduction, could increase (18)F-FDG PET diagnostic sensitivity and further motivate its inclusion in the clinical assessment of AD.

This FDG-PET study with SPM99 compared 46 patients with sporadic Alzheimer disease (SAD) to 40 patients with familial AD (FAD) and to 35 matched controls. AD groups had equivalent metabolic (METglu) reductions in several cortical and limbic areas with respect to the controls. Patients with FAD showed decreased METglu in the posterior cingulate, parahippocampal, and occipital cortex as compared to the patients with SAD (p < 0.001). Genetic factors lead to phenotypic differences in AD.

Diabetes mellitus (DM) is associated with enhanced lipid oxidation and persistent platelet activation. We investigated whether oxidant stress (OS) also affects circulating proteins and is associated with an abnormal coagulative pattern. In 72 type 2 DM (T2DM) patients, urinary 8-iso-prostaglandin (PG) F2alpha and 11-dehydro-thromboxane B2 (TXM) were measured as markers of lipid peroxidation and platelet activation, respectively. The carbonyl content of plasma proteins (PCARB) was measured as global index of protein oxidation. 8-Iso-PGF2alpha and PCARB levels were higher in DM patients than in controls (P < 0.05). Likewise, both TXM and prothrombin F1+2 levels were higher in diabetics (P < 0.05). By contrast, anticoagulant markers, such as activated protein C, protein C activation peptide, and soluble thrombomodulin (TM) were depressed in T2DM (P < 0.05). In conclusion, OS in T2DM involves circulating proteins and is associated with an unbalanced promotion of procoagulant reactions. These effects in concert with platelet activation may contribute to atherothrombotic complications in T2DM.

Activation of factor VIII (FVIII) by thrombin plays a fundamental role in the amplification of the coagulation cascade and takes place through specific proteolytic cleavages at Arg(372), Arg(740) and Arg(1689). Full FVIII activation requires cleavage at Arg(372), a process involving the alpha-thrombin exosite-II; referred to as heparin-binding site (HBS). The present study was aimed at investigating the effect of glycoprotein Ibalpha (GpIbalpha; 1-282 fragment) binding to thrombin HBS on FVIII activation. Similar experiments were also performed using a synthetic peptide modelled on the 268-282 sequence of GpIbalpha, and sulphated successfully at all tyrosine residues present along its sequence, at positions 276, 278 and 279. Both GpIbalpha 1-282 and the sulphated GpIb 268-282 peptides induced a progressive decrease (up to 70%) in activated FVIII generation, assessed by coagulation and FXa-generation assays. Furthermore, SDS/PAGE and Western-blot experiments showed that the specific appearance of the 44 kDa A2 domain on cleavage of the FVIII Arg(372)-Ser(373) peptide bond was delayed significantly in the presence of either GpIbalpha 1-282 or GpIb 268-282 peptide. Moreover, the effect of the latter on thrombin-mediated hydrolysis of a peptide having the sequence 341-376 of FVIII was investigated using reverse-phase HPLC. The k (cat)/ K (m) values of the FVIII 341-376 peptide hydrolysis by thrombin decreased linearly as a function of the GpIbalpha 268-282 peptide concentration, according to a competitive inhibition effect. Taken together, these experiments suggest that the sulphated 268-282 region of GpIbalpha binds to thrombin HBS, and is responsible for the inhibition of the Arg(372)-Ser(373) bond cleavage and activation of FVIII.

The aim of this study was to investigate the effect of deep-brain stimulation of the subthalamic nucleus (STN) on regional cerebral blood flow (rCBF) throughout the entire brain volume in patients with Parkinson's disease and to evaluate which of the brain areas showing an rCBF increase during STN stimulation related significantly to the improvement in motor function.Ten consecutive Parkinson's disease patients (6 men, 4 women; mean age +/- SD, 59 +/- 8 y) with bilateral STN stimulators underwent 3 rCBF SPECT examinations at rest: the first preoperatively and the second and third postoperatively (follow-up, 4.8 +/- 1.4 mo) with STN stimulators on and off, respectively. The motor unified Parkinson's disease rating scale, the Hoehn and Yahr disability scale, and the Schwab and England activities-of-daily-living scale were used to evaluate the clinical state under each condition. Statistical parametric mapping was used to investigate rCBF during STN stimulation in comparison with rCBF preoperatively and with STN stimulators off. Also evaluated with statistical parametric mapping was the relationship between rCBF and individual motor scores used as covariates of interest.STN stimulation significantly changed rCBF in the right pre-supplementary motor area (pre-SMA), anterior cingulate cortex, and dorsolateral prefrontal cortex and in the medial Brodmann's area 8 (BA8) as defined in the atlas of Talairach and Tournoux (P0.05 corrected for multiple comparisons). The rCBF in these areas increased from the preoperative condition to the stimulators-on condition and decreased again after the stimulators were switched off. A significant correlation was detected between the improvement in motor scores and the rCBF increase only in the right pre-SMA and in the anterior cingulate motor area (P0.005, uncorrected).According to the topographic organization of the primate STN, our study shows that stimulation of the STN leads to rCBF increases in the motor (pre-SMA), associative, and limbic territories (anterior cingulate) in the frontal cortex. The significant correlation between motor improvement and rCBF increase in the pre-SMA and the anterior cingulate motor area reinforces the hypothesis that STN stimulation in parkinsonian patients can potentiate the cortical areas participating in higher-order aspects of motor control.

Immune tolerance (IT) in hemophilia A patients with anti-factor VIII antibodies is generally based on daily factor VIII administrations. Here we report the preliminary results of an immune tolerance regimen based on recombinant high-dose (400 U/kg) factor VIII boluses administered at 48-hour intervals. Two high responder hemophilia A patients aged 2 and 3 years received this treatment without the need of permanent venous access. In both cases the IT regimen caused an anamnestic response of less than three weeks' duration and an antibody reduction to less than 5 Bethesda units was achieved in about 8-10 weeks. In the child with a more prolonged follow-up the inhibitor became undetectable after four months and factor VIII recovery at 6 months was85%. This intermittent high-dose regimen seems to be effective in rapidly inducing immune tolerance and seems particularly suitable for very young children in whom it may be useful to avoid the risks and to reduce the psychological burden of permanent venous access.

The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This, study investigated the biochemical and functional effects of alpha-thrombin oxidation by MPOS. This system, in the presence of 100 microM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCI, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human alpha-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin.

We report a family with inherited macrothrombocytopenia and characteristic large membrane complexes in the platelets. Two affected subjects had platelet counts of 40 and 65 x 10(9)/L respectively as assessed by contrast phase microscopy. Ultrastructural studies revealed giant spheroid platelets with characteristic large membrane complexes and/or giant vacuoles containing platelet organelles. Immunohistochemical studies of actin and tubulin showed a disorganization of the microtubule and actin systems. These abnormalities were absent in leukocytes, indicating a platelet-specific cytoskeleton disorder. Platelet autoantibodies were repeatedly absent. Nevertheless, in the peripheral blood we observed several figures of platelet phagocytosis by macrophages and neutrophils. The in vitro aggregometric response of platelets to ADP, collagen, thrombin, ristocetin was present, but shape change was absent. The urinary excretion of thromboxane A2 metabolites of the affected subjects were approximately 2 standard deviations above control values, in spite of a reduced maximal biosynthetic capacity of thromboxane from giant platelets assessed in vitro during whole blood clotting. This inherited platelet disorder shows structural and functional features which allow to distinguish it from other syndromes associated with giant platelets. We also propose to include ultrastructural and cytoskeletal studies in the diagnosis as well as in the classification of inherited giant platelet disorders.

Background —Thrombin binds to platelet glycoprotein Ib (Gp Ib), and this interaction contributes to platelet activation. Thrombin ligation to Gp Ib was recently shown to be inhibited by heparin, thus raising the hypothesis, investigated in this article, that heparin might inhibit thrombin-induced platelet activation. Methods and Results —Aggregation of gel-filtered platelets by 1 nmol/L thrombin was reduced by both high-molecular-weight (MW) (14 500-Da) and low-MW (4500-Da) heparin, with IC 50 values of 1.65±0.26 and 5.13±0.8 μmol/L, respectively. Homogeneous-MW fractions (16 000- to 13 000-Da range) were used to evaluate the heparin effect on intracytoplasmic calcium release by thrombin. Calcium mobilization by 1 nmol/L thrombin was reduced as a function of heparin concentration, and the inhibitory effect was correlated to the MW of heparin fractions (IC 50 values were 1.9±0.39, 6.07±0.83, and 14.8±0.43 μmol/L for 16 000-, 9000-, and 3000-Da heparin, respectively). Platelet aggregation and calcium mobilization by ADP and by the thrombin receptor–activating peptide were not affected by heparin. The activation of Gp Ib–depleted platelets by α-thrombin was not inhibited by heparin. Moreover, platelet stimulation by heparin binding site phosphopyridoxylated thrombin, which has a severe impairment of Gp Ib ligation, was not affected by heparin. Finally, heparin did not interfere with the hydrolysis by thrombin of the protease-activated receptor 1. Conclusions —These results demonstrated that heparin, by inhibiting the thrombin–Gp Ib interaction, is able to interfere with thrombin-induced platelet activation. The extent of the inhibitory effect is directly related to the MW of heparin fractions.

Platelet glycoprotein Ib (GpIb) is an integral platelet membrane glycoprotein which plays a major role in haemostasis, being involved in both von Willebrand factor (vWF) and alpha-thrombin high affinity binding. Such interactions contribute to the early adhesion of platelets to exposed subendothelium and to the process of platelet activation. Glycoprotein Ib belongs to the so calledleucine rich repeat(LRR) family of proteins, characterized by a structural motif consisting of the presence of one or more tandem LRRs, flanked by conserved sequences. Several experimental strategies have recently documented the involvement of the thrombin domain referred to as 'heparin binding site' in the binding to GpIb. This review is aimed at reporting on the structural mapping of both alpha-thrombin and GpIb domains involved in such interaction and on possible roles of thrombin-GpIb binding on the mechanisms supporting the platelet activation.

A prothrombin-time-derived method was used to measure plasma fibrinogen concentration (PFC) in 286 samples from 242 normal and 44 orally anticoagulated subjects. Absorbance changes at 405 nm (deltaOD) during the clotting process were obtained by an automatic coagulometer and their relationship with plasma fibrinogen concentration (range 90-1090 mg/dl), measured by the Clauss method, was investigated. A weighted linear regression between the deltaOD and the Clauss-derived PFC values provided the best fit of the experimental data. The fitting equation was found to be reliable and accurate for PFC determination in normal subjects, whereas a systematic overestimate of fibrinogen level was demonstrated in plasma with high fibrinogen concentrations (> 400 mg/dl) and in plasma from anticoagulated patients. The systematic overestimate in the latter samples could be a result of an increased fibrin gel turbidity, as shown by in-vitro experiments using purified fibrinogen clotted by different thrombin concentrations. The PFC overestimate by the prothrombin-time-derived method could also be experimentally reproduced by competitively inhibiting thrombin-fibrinogen interaction by hirudin 54-65 peptide and the fibrinogen fragment E. A similar qualitative result was also found for the prothrombin-time-derived method in the presence of the Gly-Pro-Arg-Pro peptide, which competitively inhibits the end-to-end fibrin aggregation process. Notably, under both the above experimental conditions, the Clauss method underestimated the PFC. On the other hand, the 'clot recovery' method was minimally affected by the above inhibitors. These results indicate that the prothrombin-time-derived method is accurate and precise for most routine purposes. Its precision seems inadequate, however, under those conditions where the prothrombin time is prolonged (such as anticoagulant therapy) and in the presence of high fibrinogen levels.

Although treatments with oral anti‐vitamin K agents have become more refined and safer over the years, physicians are reluctant to prescribe these agents for fear they will cause bleeding, particular in patients with ischaemic heart disease

SummaryThe platelet membrane glycoprotein lb (Gplb) has a high affinity binding site for α-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as “heparin binding site” (HBS) and “fibrino#gen recognition site” (FRS) were investigated as the possible domains involved in Gplb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-α-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of Gplb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the other hand, the role of FRS in the thrombin-GC interaction could be excluded by other experiments showing that GC in solution could not influence the interaction of α-thrombin with two substrates which bind to both the catalytic site and the fibrinogen recognition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2) the A α-chain of fibrinogen. Altogether these results demonstrated that GC interaction with thrombin involves the enzyme heparin binding site, whereas the fibrinogen recognition site does not play a significant role.

Background IDO1 degrades tryptophan (TRP) into intermediates with immune-modulating activities and endothelium-relaxing properties, collectively referred to as kynurenines (KYN). A contributory effect of IDO1 to inflammatory autoimmune pathologies, such as rheumatoid arthritis and systemic lupus erythematosus, has been recently described. Systemic sclerosis (SSc) is an autoimmune systemic disease characterized by vascular abnormalities and diffuse fibrosis in skin and internal organs. Objectives We aimed to address whether IDO1-driven kynurenine (KYN) production correlates with degree of inflammation and clinical features in SSc patients. Methods Peripheral blood was obtained from 70 consecutive adult patients with SSc. Serum KYN and TRP levels were measured with RP-HPLC. Pro-angiogenic hepatocyte growth factor (HGF) and placental growth factor (PlGF) were quantitated with ELISA. Results Serum KYN levels were higher in SSc compared with healthy controls (2.55±1.47 μM versus 1.74±0.30 μM; p=0.018) and positively correlated with serum C-reactive protein (r=0.35, p=0.0042) and erythrocyte sedimentation rate (r=0.26, p=0.037), but not with HGF or PlGF levels, complement C3 or C4 levels, presence of skin ulcers, modified Rodnan skin score (MRSS) or disease activity score. Interestingly, KYN levels positively correlated with pulmonary arterial pressure (r=0.38; p=0.017). However, KYN levels were superimposable in patients with normal chest CT findings and in those with clinical and radiographic evidence of alveolitis or lung fibrosis. Conclusions The generation of KYN is heightened in SSc patients and it could be related to the activation of counter-regulatory mechanisms of both inflammation (related to the production of pro-inflammatory cytokines) and vasoconstriction. The role played by KYN in promoting autoimmune circuits in SSc remains to be elucidated. Disclosure of Interest None Declared

Sepsis causes widespread microvascular injury and thrombosis. Some hemostatic factors mediate the mechanisms involved in sepsis-related organ ischemia and failure. Oxidative stress is also increased in sepsis and reactive oxygen species (ROS) favor secretion of von Willebrand factor (vWF) multimers from endothelium and inhibit vWF proteolysis by ADAMTS-13. Moreover, the enzyme indoleamine-2,3-dioxygenase, an important immune regulator, is activated in sepsis and, through generation of kynurenins, promotes antioxidative and anti-infective activities. This study evaluated the relative role of ADAMTS-13, vWF and fibrinogen in the morbidity and mortality of patients with septic shock (SS). The above hemostatic factors were measured together with kynurenine and plasma protein carbonyls, marker of oxidative stress.