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In cirrhosis, decreased portal flow velocity, thrombophilia factors, and portal hypertension are considered risk factors for portal vein thrombosis (PVT). In cirrhosis, the transformation of the stellate cells causes a progressive decrease of ADAMTS-13, while VWF multimers secretion by endothelial cells is strongly enhanced. This imbalance leads to an accumulation of ultra-large VWF multimers that in sinusoidal circulation could favor PVT both in intra- and extra-hepatic branches, mostly in decompensated cirrhosis. This prospective study was aimed at identifying possible clinical, biochemical, and hemostatic factors predictive for non-tumoral PVT in a cohort of patients with compensated cirrhosis.Seventynine compensated cirrhosis patients were prospectively followed for 48 months, receiving a periodic Doppler-ultrasound liver examination associated with an extensive evaluation of clinical, biochemical, and hemostatic profile.Five patients developed PVT (cumulative prevalence = 6.3%), occurring 4-36 months after enrollment. In logistic regression analysis, the ADAMTS-13/VWF:GpIbR ratio 0.4 was the only independent variable significantly associated with PVT (OR 14.6, 95% C.I.:1.36-157.2, p = 0.027). A Cox-regression-analysis confirmed this finding (HR = 7.7, p = 0.027).The ADAMTS-13/VWF ratio 0.4 measured in compensated cirrhosis could be a reliable predictive biomarker for PVT development, paving the way to novel therapeutic strategies to prevent and treat PVT in this clinical setting.

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy caused by a hereditary or immune-mediated deficiency of the enzyme ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). TTPs are caused by the following pathophysiological mechanisms: (1) the presence of inhibitory autoantibodies against ADAMTS13; and (2) hereditary mutations of the ADAMTS13 gene, which is present on chromosome 9. In both syndromes, TTP results from a severe deficiency of ADAMTS13, which is responsible for the impaired proteolytic processing of high-molecular-weight von Willebrand factor (HMW-VWF) multimers, which avidly interact with platelets and subendothelial collagen and promote tissue and multiorgan ischemia. Although the acute presentation of the occurring symptoms in acquired and hereditary TTPs is similar (microangiopathic hemolytic anemia, thrombocytopenia, and variable ischemic end-organ injury), their intensity, incidence, and precipitating factors are different, although, in both forms, a severe ADAMTS13 deficiency characterizes their physiopathology. This review is aimed at exploring the possible factors responsible for the different clinical and pathological features occurring in hereditary and immune-mediated TTPs.

Extensive data support the superior safety without any trade-off in efficacy of direct oral anticoagulants (DOACs) compared to vitamin K antagonists (VKA) in patients with nonvalvular atrial fibrillation, deep venous thrombosis or pulmonary embolism. Whether DOACs may be successfully used to treat complex and fragile patients with percutaneous endoscopic gastrostomy (PEG) remains to be proven. The purpose of this pilot study was to evaluate the feasibility, anticoagulant effect, and preliminary safety/efficacy profile of edoxaban administered via PEG in patients with an indication for long-term oral anticoagulation.In this prospective, single-arm, pilot study, 12 patients with PEG and guideline-recommended indication for anticoagulation for nonvalvular atrial fibrillation were prospectively enrolled. Crushed edoxaban at approved doses was administered via PEG. Quantitative measures of edoxaban's anti-factor Xa activity were performed at steady state. Thromboembolic and bleeding events were assessed at one-month follow-up.Steady state edoxaban plasma levels were at therapeutic range in all patients; mean plasma concentration was 208.5 (±78.6) ng/ml. At one month follow-up, none had suffered a thromboembolic event; one developed minor bleeding, and one died from non-cardiovascular death, owing to sudden worsening of a pre-existing underlying severe condition.In this pilot investigation, we report for the first time that crushed edoxaban, administered at approved doses through PEG in fragile and complex patients, is feasible, results in therapeutic edoxaban concentrations, and is apparently effective and safe.

The index case is a 21-year-old Italian woman with a mild hemorrhagic syndrome and von Willebrand factor antigen (VWF:Ag) = 34.3 U/dl, VWF recombinant glycoprotein Ib (VWF:GpIbR) = 32.8 U/dl, and factor VIII (FVIII) = 55.3 IU/dl.The aim of this study is to characterize from a genetic and biochemical standpoint this low VWF phenotype.Coagulation and biochemical methods were used to study the structural and functional pattern of VWF multimers in the index case's plasma. Recombinant wild-type and p.P1127S VWF variants were produced using human embryonic kidney (HEK)-293 cells. In addition, genetic screening was carried out to detect single nucleotide variants of some scavenger VWF/FVIII receptor genes such as CLEC4M, STAB2, and ASGR2.Genetic investigation revealed that the index case inherited from her mother the heterozygous missense mutation c.3379C T (VWF exon 25), causing the p.P1127S substitution in the VWF D'D3 domain. The index case was also homozygous for the scavenger receptor ASGR2 c.-95 CC-genotype. Desmopressin normalized the VWF level of the patient, although its clearance was faster (tThe p.P1127S variant may cause a low VWF phenotype, stemming from an increased VWF affinity for the scavenger receptor LRP1 and, consequently, an accelerated clearance of VWF.

Von Willebrand factor (VWF), a blood multimeric protein with a very high molecular weight, plays a crucial role in the primary haemostasis, the physiological process characterized by the adhesion of blood platelets to the injured vessel wall. Hydrodynamic forces are responsible for extensive conformational transitions in the VWF multimers that change their structure from a globular form to a stretched linear conformation. This feature makes this protein particularly prone to be investigated by mechanochemistry, the branch of the biophysical chemistry devoted to investigating the effects of shear forces on protein conformation. This review describes the structural elements of the VWF molecule involved in the biochemical response to shear forces. The stretched VWF conformation favors the interaction with the platelet GpIb and at the same time with ADAMTS-13, the zinc-protease that cleaves VWF in the A2 domain, limiting its prothrombotic capacity. The shear-induced conformational transitions favor also a process of self-aggregation, responsible for the formation of a spider-web like network, particularly efficient in the trapping process of flowing platelets. The investigation of the biophysical effects of shear forces on VWF conformation contributes to unraveling the molecular mechanisms of many types of thrombotic and haemorrhagic syndromes.

Background Recombinant (rec-) coagulation factor VIII concentrates available for hemophilia A (HA) treatment differ in cell line production and structure, which could affect their pharmacodynamics and immunogenicity. Clinical trials showed that previously untreated patients with severe HA present higher rates of inhibitor development if treated with rec-FVIII products and that differences do exist as to inhibitor's formation among different rec-FVIII products. This finding could arise from several causes, such as absence of von Willebrand factor, different glycosylation profiles, or processes of molecular aggregation of the recombinant FVIII molecules. Objectives/Methods In this study, using size exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS) spectroscopy, and functional biochemical assays, we investigated the purity grade, FX activating ability, and aggregation status of three recombinant marketed products (Advate [Baxalta], Refacto AF [Pfizer], and Kogenate [Bayer]). Results The overall analysis of the results obtained with SE-HPLC and DLS spectroscopy showed that the three recombinant FVIII concentrates contain low but significant amounts of molecular aggregates. This phenomenon was less evident for the Advate product. Molecular aggregation negatively affects the in vitro pharmacodynamics of the concentrates with higher aggregates' content. Conclusions This study shows that the three pharmaceutical formulations of recombinant FVIII contain variable amounts of molecular aggregates after their reconstitution at therapeutic concentrations. This phenomenon negatively affects the in vitro potency of the products with higher aggregates' content and might be invoked as a contributing cause of their increased risk to induce the formation of FVIII inhibitors.

Current guidelines recommend caution in prescribing concomitant use of direct-acting oral anticoagulants (DOACs) and antiepileptic drugs due to drug-drug interactions leading to potential risk of DOACs subtherapeutic concentration and treatment failure. Herein we report a significant interaction between carbamazepine (CZP) and apixaban, causing subtherapeutic concentration of the drug in a patient with atrial fibrillation who had a transient ischemic attack (TIA) episode. Another anti-Xa DOAC, edoxaban, administered to the patient after TIA occurrence did not show significant interaction with CZP. In addition to confirm that cautions should be used when antiepileptic and DOACs are concomitantly prescribed, the present case also demonstrates that, in the management of certain subsets of patients who need anticoagulant treatment, measurement of DOAC plasma concentration can help guide a personalized management and avoid adverse clinical outcomes.

Patients with novel coronavirus pneumonia show increased thrombotic risk. Although hemostatic alterations have been described in novel coronavirus pneumonia patients, case-control studies of von Willebrand factor (VWF), factor VIII (FVIII), and a disintegrin-like and metalloprotease with thrombospondin type I motif, member 13 (ADAMTS13) are lacking. VWF, ADAMTS13, FVIII, D-dimer, C-reactive protein, and routine blood cells and chemistry were measured in 10 novel coronavirus pneumonia patients and 10 non-novel coronavirus pneumonia controls. Hemostatic factors were measured less than 48 h of hospital admission in patients without invasive ventilation. D-Dimer, C-reactive protein, and fibrinogen concentrations, high in both groups, did not differ significantly in novel coronavirus pneumonia vs. non-novel coronavirus pneumonia patients. Median VWF-antigen (324 vs. 153 IU/dl, P 

Direct oral anticoagulants (DOAC) and vitamin K antagonist drugs (VKA) are recommended for stroke prevention in atrial fibrillation and for treatment of venous thromboembolism. Undoubtedly, DOAC have contributed to improve quality of life of these patients, but unfortunately, available 'real world' data show a very high variable compliance to DOAC.to evaluate predictors that adversely affect therapeutic adherence in patients naivethis study was conducted on an outpatient population in oral anticoagulant therapy in a period between January 2019 and February 2020. Patients naiveto DOAC and treated for at least 6 months were enrolled. Non-Italian-speaking patients, cognitive or psychiatric disorders, refusal to participate or non-consent to the interview were exclusion criteria. A socio-demographic scale and the 8-item Morisky scale (MMAS-8) questionnaire assessed therapeutic adherence.One hundred two DOAC-naïve patients were selected from a population of 407 patients on the first visit at our centre. The population was homogeneously represented for gender (males 48%). The mean age was 79.5 years. Atrial fibrillation (65.7%) resulted the main reason for DOAC prescription and a polypharmacy was detected in 47.1% of the patients. Moreover, an optimal adherence to DOAC therapy was assessed in less than 30% of patients.Polypharmacy, patient's isolation, such as a low education level were statistically associated with a low therapeutic adherence. Therapeutic adherence remains an unsolved problem for anticoagulated patient. To identify patients at higher risk of poor compliance and therapeutic failure and establish targeted care pathways is a priority.

Background Acquired von Willebrand factor (VWF) deficiency was described in Philadelphia‐negative myeloproliferative neoplasms, especially in essential thrombocythemia (ET). VWF phenotype in contemporary patients with polycythemia vera (PV) remains less explored. Objectives To characterize the VWF phenotype in PV and to compare VWF phenotype in PV with matched healthy subjects and ET patients. Patients/Methods We studied 48 PV patients, treated according to current recommendations (hematocrit ≤ 45%, on low‐dose aspirin prophylaxis); 48 healthy and 41 subjects with ET, all sex, age, and blood group matched. We measured VWF antigen, activity, multimeric pattern, ADAMTS‐13, and factor VIII (FVIII) antigen. Results In patients with PV, VWF antigen and activity were significantly higher than in healthy subjects (antigen: 119[96‐137] vs 93[79‐107] IU/dL; activity: 114[95‐128] vs 90[79‐107] IU/dL, respectively, medians and interquartile, P

We observed a 55-year-old Italian man who presented with mucosal and cutaneous bleeding. Results of his blood analysis showed low levels of von Willebrand factor (VWF) antigen and VWF activity (both VWF ristocetin cofactor and VWF collagen binding), mild thrombocytopenia, increased ristocetin-induced platelet aggregation, and a deficiency of high-molecular-weight multimers, all typical phenotypic hallmarks of type 2B von Willebrand disease (VWD). The analysis of the VWF gene sequence revealed heterozygous in cis mutations: (1) c.2771G>A and (2) c.6532G>T substitutions in the exons 21 and 37, respectively. The first mutation causes the substitution of an Arg residue with a Gln at position 924, in the D′D3 domain. The second mutation causes an Ala to Ser substitution at position 2178 in the D4 domain. The patient’s daughter did not present the same fatherly mutations but showed only the heterozygous polymorphic c.3379C>T mutation in exon 25 of the VWF gene causing the p.P1127S substitution, inherited from her mother. The in vitro expression of the heterozygous in cis VWF mutant rVWFWT/rVWF924Q-2178S confirmed and recapitulated the ex vivo VWF findings. Molecular modeling showed that these in cis mutations stabilize a partially stretched and open conformation of the VWF monomer. Transmission electron microscopy and atomic force microscopy showed in the heterozygous recombinant form rVWFWT/rVWF924Q-2178S a stretched conformation, forming strings even under static conditions. Thus, the heterozygous in cis mutations 924Q/2178S promote conformational transitions in the VWF molecule, causing a type 2B–like VWD phenotype, despite the absence of typical mutations in the A1 domain of VWF.

Extensive data support the safety of direct oral anticoagulants compared with vitamin K antagonists in patients with non‐valvular atrial fibrillation, leading to a significantly increase in the use of these compounds in clinical practice. However, there is no compelling evidence supporting the use of direct oral anticoagulant in individuals who are intubated or have a percutaneous endoscopic gastrostomy (PEG): patients with several co‐morbidities are underrepresented in clinical trials, so the best long‐term strategy for anticoagulation is difficult to ascertain. The aim of the present report was to evaluate the safety and efficacy of edoxaban administered via PEG in a patient with heart failure and a history of atrial fibrillation affected by amyotrophic lateral sclerosis (ALS). A 71‐year‐old man with atrial fibrillation, advanced ALS, type II diabetes mellitus, and hypertension presented to the emergency department with dyspnoea and tachycardia. Because vitamin K antagonist and rivaroxaban 15 mg were dropped because of difficult international normalized ratio control (time in therapeutic range

Portal vein thrombosis (PVT) dramatically changes the prognosis of cirrhotic patients, especially those waiting for liver transplantation. However, the possible contribution to PVT of von Willebrand factor (VWF) and ADAMTS-13 is poorly documented. The aim of our study was to assess the presence of alterations of VWF and ADAMTS-13 serum levels in cirrhotic patients with PVT. Twenty-four patients with PVT (group PVT) and 60 without PVT (group without PVT) were enrolled. A comprehensive analysis of biochemical and hemostatic parameters was performed. ADAMTS-13 activity was significantly lower in group A (median 16.8 vs. 69.1 %, p = 0.0047). Group PVT, compared to group without PVT, showed a significantly higher VWF:act, (median 308.4 vs 203.3 %, p = 0.032), whereas no difference was observed for VWF:Ag, FVIII level and the presence of risk factors for venous thromboembolism. No correlation was found between the Child-Pugh score and ADAMTS-13 activity. In multivariable logistic regression analysis performed on data concerning both group PVT and without PVT, only the ADAMTS-13 activity (p = 0.007) was independently and inversely associated with PVT. In conclusion, ADAMTS-13 activity is independently associated with PVT in cirrhotic patients.

The direct oral anticoagulant apixaban (APX), a strong factor Xa inhibitor, binds also to plasma proteins, especially albumin, and minimally to α1-acid glycoprotein. Although APX can cross the red cell membrane, due to its chemical structure, and could bind to haemoglobin (Hb), no investigation was performed on this possible phenomenon that could affect the APX plasma concentration and thus its pharmacokinetics and pharmacodynamics. We addressed this issue by (1) measuring the levels of APX and haematological/biochemical parameters in 90 patients on APX therapy; (2) assessing the effect of APX on oxygen saturation curves of Hb; (3) testing the direct APX binding to Hb by fluorescence spectroscopy and a zinc-induced precipitation of Hb coupled to a reversed-phase high-performance liquid chromatography (HPLC)-based method; and (4) simulating in silico by molecular docking the APX interaction with human Hb. In a multivariable analysis, Hb was the only independent variable significantly and inversely associated in 90 patients with APX peak plasma level, at variance with patients treated with rivaroxaban (n = 86) and dabigatran (n = 34) therapy. APX causes a progressive left-shift of the oxygen dissociation curve of purified Hb solution, with a Kd ≅300 µM. Fluorescence- and HPLC-based assays concordantly showed that APX binds to Hb with a Kd ≅350 µM. Finally, docking simulations showed that APX can fit into in the central cavity of Hb. These findings support the hypothesis that APX does bind to Hb, which, due to its millimolar concentration in blood, can act as ‘buffer’ for the drug and consequently affect its free plasma level.

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

Background : Double filtration plasmapheresis (DFPP) is a form of therapeutic plasma exchange (TPE) that removes high-molecular-weight (HMW) pathological mediators from the plasma with two filters: a plasma separator and a plasma fractionator. Relative to simple TPE, the semi-selectiveness of DFPP reduces protein loss and the need for substitution fluid. However, depending on the choice of plasma fractionator, DFPP may negatively impact hemostatic components such as HMW coagulation factors. Aim: To determine the impact of DFPP on various hemostatic parameters (i.e. FVIII activity, fibrinogen level, vWF level and activity, ADAMTS13 level and activity, protein C (PC) activity, protein S (PS) activity, antithrombin (AT) activity, prothrombin time (PT), and activated partial thromboplastin time (aPTT)). Methods: Fourteen patients undergoing weekly, bimonthly, or monthly DFPP sessions for hematologic conditions (n=11) or nervous system disorders (n=3) were recruited. Hemostatic parameters were measured immediately before and after 27 DFPP sessions (1-4 sessions/patient). The treatment volume was standardized at one plasma volume, and anticoagulation was performed with ACD-A citrate dextrose solution. EC-30W and EC-50W were used as the plasma fractionators in 4 and 23 sessions, respectively. In addition to primary data collection, we systematically searched PubMed, MEDLINE, and EMBASE for studies that investigated the impact of DFPP on hemostasis. No restriction was placed on filter choice. Results: In our cohort of 14 patients, all hemostatic changes were statistically significant. After a routine DFPP session, the level and/or activity of HMW proteins (>100kDa: FVIII, fibrinogen, vWF, ADAMTS13) were decreased more than those of low-molecular-weight (LMW) proteins ( Our systematic review included 26 cohort studies, 6 case reports, and 1 randomized controlled trial. Increases in INR and aPTT following DFPP were considerably higher in previous studies, which may relate to the commonality of heparin-induced anticoagulation. The present study is the first comprehensive investigation of the impact of DFPP on hemostatic parameters with such a large sample size. It is also the first study of its nature to measure ADAMTS13-related parameters. Conclusions: Routine DFPP resulted in significant reduction across all investigated hemostatic proteins and significant prolongation of clotting tests. The activities of HMW coagulation-related proteins were decreased more than those of LMW anticoagulation-related proteins. This finding suggests that DFPP may increase overall bleeding risk, a consideration for patient safety that may be of importance in continuous DFPP treatment. Additional high quality evidence is needed to elucidate the effect of DFPP on the hemostatic system. Disclosures Matino: Sanofi: Honoraria; Sobi: Honoraria, Research Funding; Roche: Research Funding; Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bioviiix: Honoraria.

SummaryCongenital thrombotic thrombocytopenic purpura (TTP) is a rare form of thrombotic microangiopathy, inherited with autosomal recessive mode as a dysfunction or severe deficiency of ADAMTS-13 (A Disinte-grin And Metalloprotease with ThromboSpondin 1 repeats Nr. 13), caused by mutations in the ADAMTS-13 gene. About 100 mutations of the ADAMTS-13 gene were identified so far, although only a few char-acterised by in vitro expression studies. A new Asp to Gly homozygous mutation at position 173 of ADAMTS-13 sequence was identified in a family of Romanian origin, with some members affected by clinical signs of TTP. In two male sons, this mutation caused a severe (< 3 %) deficiency of ADAMTS-13 activity and antigen level, associated with periodic thrombocytopenia, haemolytic anaemia and mild mental confusion. Both parents, who are cousins, showed the same mutation in heterozygous form. Expression studies of the mutant ADAMTS-13, performed in HEK293 cells, showed a severe decrease of the enzyme’s activity and secretion, although the protease was detected inside the cells. Molecular dynamics found that in the D173G mutant the interface area between the metalloprotease domain and the disintegrin-like domain significantly decreases during the simulations, while the proline-rich 20 residues linker region (LR, 285–304) between them undergoes extensive conformational changes. Inter-domain contacts are also significantly less conserved in the mutant compared to the wild-type. Both a decrease of the inter-domain contacts along with a substantial conformational rearrangement of LR interfere with the proper maturation and folding of the mutant ADAMTS-13, thus impairing its secretion.Supplementary Material to this article is available online at www.thrombosis-online.com.

Activated human platelets synthesize prostaglandin (PG) E(2), although at lower rate than thromboxane A(2). PGE(2) acts through different receptors (EP1-4), but its role in human platelet function remains poorly characterized compared with thromboxane. We studied the effect of PGE(2) and its analogs on in vitro human platelet function and platelet and megakaryocyte EP expression. Platelets preincubated with PGE(2) or its analogs were stimulated with agonists and studied by optical aggregometry. Intraplatelet calcium mobilization was investigated by the stopped flow method; platelet vasodilator-stimulated phosphoprotein (VASP), P-selectin, and microaggregates were investigated by flow cytometry. PGE(2) at nanomolar concentrations dose-dependently increased the slope (velocity) of the secondary phase of ADP-induced platelet aggregation (EC(50), 25.6 ± 6 nM; E(max) of 100 ± 19% increase versus vehicle-treated), without affecting final maximal aggregation. PGE(2) stabilized reversible aggregation induced by low ADP concentrations (EC(50), 37.7 ± 9 nM). The EP3 agonists, 11-deoxy-16,16-dimethyl PGE(2) (11d-16dm PGE(2)) and sulprostone enhanced the secondary wave of ADP-induced aggregation, with EC(50) of 48.6 ± 10 nM (E(max), 252 ± 51%) and 5 ± 2 nM (E(max), 300 ± 35%), respectively. The EP2 agonist butaprost inhibited ADP-induced secondary phase slopes (IC(50), 40 ± 20 nM). EP4 stimulation had minor inhibitory effects. 11d-16dm PGE(2) alone raised intraplatelet Ca(2+) and enhanced ADP-induced Ca(2+) increase. 11d-16dm PGE(2) and 17-phenyltrinor PGE(2) (EP3 > EP1 agonist) at nanomolar concentrations counteracted PGE(1)-induced VASP phosphorylation and induced platelet microaggregates and P-selectin expression. EP1, EP2, EP3, and EP4 were expressed on human platelets and megakaryocytes. PGE(2) through different EPs finely modulates human platelet responsiveness. These findings should inform the rational selection of novel antithrombotic strategies based on EP modulation.

Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in thrombosis and hemostasis, present in circulating blood and in secretory granules of endothelial cells and platelets. High shear stress triggers conformational changes responsible for both binding to the platelet receptor glycoprotein GpIb and its self-association, thus supporting the formation of platelet plug under flow. Ristocetin also promotes the interaction of VWF with GpIb and is able to induce platelet aggregation, and thus is largely used to mimic this effect in vitro. In this research paper, we followed the time course of VWF self-association in solution induced by ristocetin binding by light scattering and at the same time we collected atomic force microscopy images to clarify the nature of the assembly that is formed. In fact, this process evolves initially through the formation of fibrils that subsequently interact to form supramolecular structures whose dimensions would be capable of trapping platelets even in the absence of any degree of shear stress or interaction with external surfaces. This intrinsic property, that is the ability to self-aggregate, may be involved in some pathological settings that have been revealed in clinical practice.

Inflammatory lung disease is a primary cause of morbidity and mortality in cystic fibrosis (CF). Mechanisms of unresolved acute inflammation in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in nonrespiratory cells is emerging. Here we examined CFTR expression and function in human platelets (PLTs) and found that they express a biologically active CFTR. CFTR blockade gave an ∼50% reduction in lipoxin A(4) (LXA(4)) formation during PLT/polymorphonuclear leukocytes (PMN) coincubations by inhibiting the lipoxin synthase activity of PLT 12-lipoxygenase. PLTs from CF patients generated ∼40% less LXA(4) compared to healthy subject PLTs. CFTR inhibition increased PLT-dependent PMN viability (33.0±5.7 vs. 61.2±8.2%; P=0.033), suppressed nitric oxide generation (0.23±0.04 vs. 0.11±0.002 pmol/10(8) PLTs; P=0.004), while reducing AKT (1.02±0.12 vs. 0.71±0.007 U; P=0.04), and increasing p38 MAPK phosphorylation (0.650±0.09 vs. 1.04±0.24 U; P=0.03). Taken together, these findings indicate that PLTs from CF patients are affected by the molecular defect of CFTR. Moreover, this CF PLT abnormality may explain the failure of resolution in CF.

An enhanced formation of reactive oxygen species and peroxynitrite occurs in several clinical settings including diabetes, coronary artery disease, stroke, sepsis, and chronic inflammatory diseases. Peroxynitrite oxidizes methionine and tyrosine residues to methionine sulfoxide (MetSO) and 3-nitrotyrosine (NT), respectively. Notably, ADAMTS-13 cleaves von Willebrand factor (VWF) exclusively at the Tyr1605-Met1606 peptide bond in the A2 domain. We hypothesized that peroxynitrite could oxidize either or both of these amino acid residues, thus potentially affecting ADAMTS-13-mediated cleavage. We tested our hypothesis using synthetic peptide substrates based on: (1) VWF Asp1596-Ala1669 sequence (VWF74) and (2) VWF Asp1596-Ala1669 sequence containing nitrotyrosine (VWF74-NT) or methionine sulfoxide (VWF74-MetSO) at position 1605 or 1606, respectively. The peptides were treated with recombinant ADAMTS-13 and the cleavage products analyzed by RP-HPLC. VWF74 oxidized by peroxynitrite underwent a severe impairment of its hydrolysis. Likewise, VWF74-MetSO was minimally hydrolyzed, whereas VWF74-NT was hydrolyzed slightly more efficiently than VWF74. Oxidation by peroxynitrite of purified VWF multimers inhibited ADAMTS-13 hydrolysis, but did not alter their electrophoretic pattern nor their ability to induce platelet agglutination by ristocetin. Moreover, VWF purified from type 2 diabetic patients showed oxidative damage, as revealed by enhanced carbonyl, NT, and MetSO content and was partially resistant to ADAMTS-13 hydrolysis. In conclusion, peroxynitrite may contribute to prothrombotic effects, hindering the proteolytic processing by ADAMTS-13 of high-molecular-weight VWF multimers, which have the highest ability to bind and activate platelets in the microcirculation.

Prothrombin deficiency is among the rarest inherited coagulation disorders, with a prevalence of approximately 1:2,000,000. Two main phenotypes can be distinguished: (1) hypoprothrombinemia (type I deficiency), characterized by concomitantly low levels of activity and antigen; and (2) dysprothrombinemia (type II deficiency), characterized by the normal or near-normal synthesis of a dysfunctional protein. In some cases, hypoprothrombinemia associated with dysprothrombinemia was also described in compound heterozygous defects. No living patient with undetectable plasma prothrombin has been reported to date. Prothrombin is encoded by a gene of approximately 21 kb located on chromosome 11 and containing 14 exons. Forty different mutations have been identified and characterized in prothrombin deficiency. Many of them surround the catalytic site, whereas another "hot spot" is localized in the recognition domain called anion binding exosite I, also called fibrinogen recognition site. Recently, mutations were identified also in the Na (+)-binding loop and in the light A-chain of thrombin. Most hypoprothrombinemia-associated mutations are missense, but there are also nonsense mutations leading to stop codons and one single nucleotide deletion. Finally, the main aspects of clinical manifestations and therapy of congenital prothrombin deficiency are presented and discussed.

Summary Objective The aim of the study was to evaluate the effects of an intensive rehabilitation programme on thyroid metabolism, the relationship between disability and thyroid hormone level, and the occurrence of nonthyroidal illness syndrome (NTIS) before and after rehabilitation. Design, subjects and measurements This was a clinical prospective study. Orthopaedic surgery patients (n = 82) were classified into two groups: patients in whom early active mobilization and walking were possible (walking group, WG, n = 45), and patients in whom these were not recommended (nonwalking group, NWG, n = 37). Levels of free T3 (fT3), fT4, TSH and rT3 were measured before and after surgery, and then at 1, 3, 7, 14 and 30 days from the beginning of rehabilitation. Personal, nutritional and clinical data were acquired for all patients. The Barthel Index (BI) was used to assess disability before and after rehabilitation. Results Immediately after surgery, both groups of patients showed a significant decrease in mean fT3 concentrations and a significant increase in rT3; mean fT4 values decreased significantly only in NWG patients. Once rehabilitation had been completed, fT3 and rT3 levels returned to baseline values in WG patients. In NWG patients mean fT3 and fT4 levels continued to decrease significantly and rT3 values remained significantly high until the end of rehabilitation. NTIS occurred in 38% of the NWG patients. No significant changes in TSH levels were observed in either group. Finally, we observed a direct correlation between fT3 levels and the BI in WG patients. Conclusions Our data suggest that early patient mobilization and physical activity during an active and intensive rehabilitation programme induce recovery of thyroid function and avoid occurrence of NTIS.

Gliotoxin (GT) belongs to the epipolythiodioxopiperazine class of toxins secreted from certain fungi including Aspergillus fumigatus, which is the most prolific producer of this secondary metabolite. Recently, enhanced amounts of GT were found in in vitro biofilm-grown A. fumigatus mycelium. To further correlate the A. fumigatus biofilm growth phenotype with the enhanced secretion of GT, a polyclonal antibody (pAb) was produced by immunizing mice against GT. By an indirect immunofluorescent assay, pAb was then able to recognize specifically GT onto A. fumigatus Af293 biofilm formed on human pulmonary epithelial cells. Then, treating Af293 biofilms with a compound which reduces the GT disulfide bonds provoked shutdown of the GT-specific immunofluorescence (IF) signals along the hyphae. To explore the potential of GT for diagnostic use, pAb was shown to react with GT on hyphae into Aspergillus culture-positive respiratory tract specimens from patients with probable invasive aspergillosis (IA) and into tissue specimens from the lungs of patients with proven IA. As the presence of fungal hyphae in clinical specimens strongly indicates the in vivo A. fumigatus growth as a biofilm, anti-GT antibodies could be a specific and sensitive diagnostic tool for detecting A. fumigatus biofilm-associated clinical infections.

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.

Prothrombin (factor II [FII]) deficiency is a rare inherited coagulation disorder, having a prevalence of approximately 1 in 2,000,000. Two phenotypes can be distinguished: (1) true hypoprothrombinemia (type I deficiency), characterized by concomitantly low levels of the zymogen antigen; and (2) dysprothrombinemia (type II deficiency), characterized by the normal or near-normal synthesis of a dysfunctional protein. In the latter case, recent studies showed that particular mutations in the catalytic domain of active thrombin can even impair the enzyme interaction with antithrombin, favoring thromboembolic diseases. In some cases, hypoprothrombinemia associated with dysprothrombinemia was also described in compound heterozygous defects. Prothrombin is essential for the development of mammalian organisms. No living patient with undetectable plasma prothrombin has been reported to date. Prothrombin is encoded by a ≈21 kb gene located on chromosome 11 and containing 14 exons. Thirty-nine different mutations have been identified and characterized in prothrombin deficiency. Many of these are present in the catalytic site, whereas some involve regulatory domains, such as the anion-binding exosite I, the Na+-binding loop, and the light A-chain. Most hypoprothrombinemia-associated mutations are missense, but nonsense mutations leading to stop codons and one single nucleotide deletion have also been identified. Finally, recent developments in the therapy of congenital prothrombin deficiency are presented and discussed.

The thrombogenic activity of Von Willebrand factor (VWF) is proportional to its molecular size and inversely related to its proteolysis by ADAMTS-13. Oxidation of VWF severely impairs its proteolysis by the metalloprotease. This study was aimed at assessing in patients with type 1 and type 2 diabetes whether protein carbonyls, marker of oxidative stress, are associated with both the level and oxidation status of VWF as well as with micro- and macroangiopathic complications. Eighty-three diabetic patients (41 type 1 and 42 type 2 diabetic subjects) and their respective eighty-three healthy controls were studied after verifying the availability, through institutional databases, of clinical biochemistry records spanning at least 3 years. VWF and protein carbonyls were measured by immunoassays, whereas VWF multimers were studied by SDS-agarose gel electrophoresis. Type 2 diabetic subjects had higher levels of VWF antigen (VWF:ag), VWF activity (VWF:act) and plasma proteins’ carbonyls compared to both their controls and type 1 diabetic subjects. Moreover, high molecular weight VWF multimers and specific VWF-bound carbonyls were significantly increased in subjects with micro- and macro-angiopathic complications. In both type 1 and type 2 diabetic subjects, ADAMTS-13 activity was in the normal range. In a multivariable analysis, only VWF-bound carbonyls were significantly associated with any form of thrombotic angiopathy in the entire group of T1- and T2 diabetic patients. These data provide first evidence that not only high VWF levels but also its oxidation status and the presence of high molecular weight VWF multimers that are not observed in SDS-agarose gel electrophoresis of normal subjects are associated with thrombotic angiopathies in diabetes mellitus. These findings may help identify diabetic patients particularly at risk for these complications and elucidate a new pathophysiological mechanism of thrombotic angiopathies in this clinical setting.

Microangiopathy is a term which describes a disease in the small blood vessels of the circulatory tree. Thrombotic Microangiopathy (TMA) was a term firstly introduced by Simmers in 1952 which combined several related disorders that are associated with thrombus formation in distinct organs [1]. In this group of rare diseases, the pathologic presentation is dominated by coagulation disturbances and endothelial cell injury resulting in swelling or detachment of the endothelial cell from the basement membrane, intraluminal aggregation of platelets and mechanical injury to red blood cells leading to thrombocytopenia and organ ischemia [2, 3] (see Table 1). These features are really common to various disorders, however Thrombotic Thrombocytopenic Purpura (TTP) and the Hemolytic-Uremic Syndrome (HUS), represent the major and more investigated forms whose pathogenesis has been clarified only in the last three decades [2-4]. Both TTP and HUS occur at a frequency of approximately 1-6 cases per one million people, may affect children and adults, and may each have several distinct subtypes with overlapping symptoms, but caused by differing pathophysiologic mechanisms [5].

CKD (chronic kidney disease) is a life-threatening pathology, often requiring HD (haemodialysis) and characterized by high OS (oxidative stress), inflammation and perturbation of vascular endothelium. HD patients have increased levels of vWF (von Willebrand factor), a large protein (~240 kDa) released as UL-vWF (ultra large-vWF polymers, molecular mass ~20000–50000 kDa) from vascular endothelial cells and megakaryocytes, and responsible for the initiation of primary haemostasis. The pro-haemostatic potential of vWF increases with its length, which is proteolytically regulated by ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), a zinc-protease cleaving vWF at the single Tyr1605–Met1606 bond, and by LSPs (leucocyte serine proteases), released by activated PMNs (polymorphonuclear cells) during bacterial infections. Previous studies have shown that in vitro oxidation of Met1606 hinders vWF cleavage by ADAMTS-13, resulting in the accumulation of UL-vWF that are not only more pro-thrombotic than shorter vWF oligomers, but also more efficient in binding to bacterial adhesins during sepsis. Notably, HD patients have increased risk of developing dramatic cardiovascular and septic complications, whose underlying mechanisms are largely unknown. In the present study, we first purified vWF from HD patients and then chemically characterized its oxidative state. Interestingly, HD-vWF contains high carbonyl levels and increased proportion of UL-vWF polymers that are also more resistant to ADAMTS-13. Using TMS (targeted MS) techniques, we estimated that HD-vWF contains >10% of Met1606 in the sulfoxide form. We conclude that oxidation of Met1606, impairing ADAMTS-13 cleavage, results in the accumulation of UL-vWF polymers, which recruit and activate platelets more efficiently and bind more tightly to bacterial adhesins, thus contributing to the development of thrombotic and septic complications in CKD. Abbreviations: ADAMTS-13, a disintegrin and metalloproteinase with thrombospondin motifs 13; a.m.u., atomic mass unit; CKD, chronic kidney disease; CVD, cardiovascular disease; DNP, dinitrophenylhydrazine; ESI–TOF, electrospray ionization–time-of-flight; ESRD, end-stage renal disease; HD, haemodialysis; HRP, horseradish peroxidase; LC-MS, liquid chromatography-MS; LSP, leucocyte serine protease; MetSO, methionine sulfoxide; MS/MS, tandem MS; NT, 3-nitrotyrosine; OS, oxidative stress; PMN, polymorphonuclear cell; PN, peroxynitrite; PTP, proteotypic peptide; RCM, reduced and carboxamidomethylated; RiCof, ristocetin cofactor; ROS, reactive oxygen species; RP-HPLC, reverse-phase-HPLC; TFA, trifluoroacetic acid; TIC, total ion current; TMS, targeted MS; T2D, Type 2 diabetes; vWF, von Willebrand factor; UL-vWF, ultra large-vWF; vWF-Ox, oxidized vWF

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 haemostatic potential of von Willebrand factor, a glycoprotein expressed by endothelial cells as ultra-large polymers (UL-vWF)(1), increases with its length, which in turn is regulated proteolytically by ADAMTS13, a zinc-metalloprotease selectively cleaving vWF at the Tyr1605-Met1606 bond. We have recently shown that in vitro oxidation of Met1606, under conditions mimicking those found in diseases characterized by high oxidative stress, severely impairs proteolysis by ADAMTS13, with a resulting pro-thrombotic effect caused by the accumulation of UL-vWF species. Conversely, Val1607Asp mutation, found in vWF from patients with type 2A von Willebrand disease, accelerates proteolysis of vWF, with a final hemorrhagic effect. Considering the physio-pathological importance of ADAMTS13-vWF interaction and the absence of experimental structural data, here we produced by homology modeling techniques a three-dimensional model of ADAMTS13 metalloprotease domain (M13). Thereafter, the vWF(1604-1607) peptide, containing the cleavable Tyr1605-Met1606 bond, was manually docked into the protease active site and the resulting model complex provided us key information for interpreting on structural grounds the variable effects that chemical modifications/mutations in vWF have on proteolysis by ADAMTS13.

Inflammation and coagulation constantly influence each other and are constantly in balance. Emerging evidence supports this statement in acute inflammatory diseases, such as sepsis, but it also seems to be very important in chronic inflammatory settings, such as inflammatory bowel disease (IBD). Patients with Crohn’s disease and ulcerative colitis have an increased risk of thromboembolic events, and several abnormalities concerning coagulation components occur in the endothelial cells of intestinal vessels, where most severe inflammatory abnormalities occur. The aims of this review are to update and classify the type of coagulation system abnormalities in IBD, and analyze the strict and delicate balance between coagulation and inflammation at the mucosal level. Recent studies on possible therapeutic applications arising from investigations on coagulation abnormalities associated with IBD pathogenesis will also be briefly presented and critically reviewed.

ADAMTS13 is a 190-kDa zinc protease encoded by a gene located on chromosome 9q34. This protease specifically hydrolyzes von Willebrand factor (VWF) multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains, including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1) repeats, a cysteine-rich domain, a spacer domain, and two CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein) domains. ADAMTS13 cleaves a single peptide bond (Tyr(1605)-Met(1606)) in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultralarge VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs). In this chapter, we (a) discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, (b) address the ongoing controversies, and (c) indicate the direction of future investigations.

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.

ADAMTS13 is a 190-kDa zinc protease encoded by a gene located on chromosome 9q34. This protease specifically hydrolyzes von Willebrand factor (VWF) multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains, including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1) repeats, a cysteine-rich domain, a spacer domain, and two CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein) domains. ADAMTS13 cleaves a single peptide bond (Tyr(1605)-Met(1606)) in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultralarge VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs). In this chapter, we (a) discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, (b) address the ongoing controversies, and (c) indicate the direction of future investigations.

The aim of the present study was to investigate the role of GV (glycaemic variability) in diabetic vascular complications and to explore the molecular pathways modulated by glycaemic ‘swings’. We developed a murine model. A total of 30 diabetic mice received once daily basal insulin administration plus two oral boluses of glucose solution (GV group, named ‘V’) and 30 diabetic mice received once daily basal insulin plus two oral boluses of saline solution (stable hyperglycaemia group, named ‘S’) for a period of 30 days. Glycaemia was measured eight times daily to detect GV. Finally, postischaemic vascularization, induced by hindlimb ischaemia 30 days after diabetes onset, was evaluated. We found that GV was significantly different between S and V groups, whereas no significant difference in the mean glycaemic values was detected. Laser Doppler perfusion imaging and histological analyses revealed that the ischaemia-induced angiogenesis was significantly impaired in V mice compared with S group, after ischaemic injury. In addition, immunostaining and Western blot analyses revealed that impaired angiogenic response in V mice occurred in association with reduced VEGF (vascular endothelial growth factor) production and decreased eNOS (endothelial nitric oxide synthase) and Akt (also called protein kinase B) phosphorylation. In conclusion, we describe a murine model of GV. GV causes an impairment of ischaemia-induced angiogenesis in diabetes, likely to be independent of changes in average blood glucose levels, and this impaired collateral vessel formation is associated with an alteration of the VEGF pathway.