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Aiming at exploring vascular components in multiple sclerosis (MS) with brain outflow disturbance, we combined transcriptome analysis in MS internal jugular vein (IJV) wall with WES in MS families with vertical transmission of disease. Main results were the differential expression in IJV wall of 16 MS-GWAS genes and of seven genes (GRIN2A, GRIN2B, IL20RB, IL26, PER3, PITX2, and PPARGC1A) not previously indicated by GWAS but encoding for proteins functionally interacting with MS candidate gene products. Strikingly, 22/23 genes have been previously associated with vascular or neuronal traits/diseases, nine encoded for transcriptional factors/regulators and six (CAMK2G, GRIN2A, GRIN2B, N1RD1, PER3, PPARGC1A) for circadian entrainment/rhythm components. Among the WES low-frequency (MAF ≤ 0.04) SNPs (n = 7) filtered in the 16 genes, the NR1D1 rs17616365 showed significantly different MAF in the Network for Italian Genomes affected cohort than in the 1000 Genome Project Tuscany samples. This pattern was also detected in five nonintronic variants (GRIN2B rs1805482, PER3 rs2640909, PPARGC1A rs2970847, rs8192678, and rs3755863) in genes coding for functional partners. Overall, the study proposes specific markers and low-frequency variants that might help (i) to understand perturbed biological processes in vascular tissues contributing to MS disease, and (ii) to characterize MS susceptibility genes for functional association with disease-pathways.

Background High plasma levels of activated Factor VII-Antithrombin complex (FVIIa-AT) have been associated with an increased risk of cardiovascular mortality in patients with stable coronary artery disease (CAD). Objectives To investigate if FVIIa-AT levels are associated with activated factor X generation (FXaG) in modified assays. Patients/methods Forty CAD patients were characterized for FVIIa-AT levels by ELISA and for FXaG in plasma. Novel fluorogenic FXaG assays, based on aptamers inhibiting thrombin and/or tissue factor pathway inhibitor (TFPI), were set up. Results FXaG correlated with FVIIa-AT levels (RAUC = 0.393, P = 0.012). The combination of thrombin inhibition and FXaG potentiation by using anti-thrombin and anti-TFPI aptamers, respectively, favors the study of time parameters. The progressive decrease in lag time from the lowest to the highest FVIIa-AT quartile was magnified by combining TFPI and thrombin inhibitory aptamers, thus supporting increased FXaG activity in the coagulation initiation phase. By exploring FXaG rates across FVIIa-AT quartiles, the largest relative differences were detectable at the early times (the highest versus the lowest quartile; 5.0-fold, P = 0.005 at 45 s; 3.5-fold, P = 0.001 at 55 s), and progressively decreased over time (2.3-fold, P = 0.002 at 75 s; 1.8-fold, P = 0.008 at 95 s; 1.6-fold, P = 0.022 at 115 s). Association between high FVIIa-AT levels and increased FXaG was independent of F7 −323 A1/A2 polymorphism influencing FVIIa-AT levels. Conclusions High FVIIa-AT plasma levels were associated with increased FXaG. Hypercoagulability features were specifically detectable in the coagulation initiation phase, which may have implications for cardiovascular risk prediction by either FVIIa-AT complex measurement or modified FXaG assays.

Background The asialoglycoprotein receptor (ASGPR) binds with high affinity factor VIII (FVIII) through its N-linked oligosaccharides. However, its contribution to the wide inter-individual variation of infused FVIII pharmacokinetics (PK) in hemophilia A (HA) is unknown. Objective To investigate the variability in FVIII PK outcomes in relation to genetic variation in the ASGR2, encoding the ASGPR2 subunit. Methods Thirty-two HA patients with FVIII:C ≤2 IU/dL underwent 66 single-dose FVIII PK studies. PK parameters were evaluated in relation to ASGR2 5′ untranslated region (5′UTR) polymorphisms, which were investigated by recombinant and white blood cell reverse transcription-polymerase chain reaction approaches. Results The 5′UTR polymorphisms determine a frequent and conserved haplotype (HT1) in a regulatory region. The HT1 homozygotes may differ in the amounts of alternatively spliced mRNA transcripts and thus ASGPR2 isoforms. Compared with the other ASGR2 genotypes, the c.-95TT homozygotes (n = 9), showed threefold longer Alpha HL (3.60 hours, 95% confidence interval: 1.44–5.76, p = 0.006), and the c.-95TC heterozygotes (n = 17) showed 25% shorter mean residence time (MRT; 18.5 hours, 15.0–22.0, p = 0.038) and 32% shorter Beta HL (13.5 hours, 10.9–16.0, p = 0.016). These differences were confirmed in patients (n = 27) undergoing PK studies (n = 54) with full-length FVIII only. In different linear regression models, the contribution of the ASGR2 genotypes remained significant after adjustment by ABO genotypes and von Willebrand factor (VWF) antigen levels, and explained 14% (MRT), 15 to 18% (Beta HL), and 22% (Alpha HL) of parameter variability. Conclusion Infused FVIII distribution was modulated by frequent ASGR2 genotypes, independently from and together with ABO and VWF antigen levels, which has potential implications for genetically tailored substitutive treatment in HA.

Abnormal levels of pyruvate and lactate were reported in multiple sclerosis (MS). We studied the response of markers of mitochondrial function to rehabilitation in relation to type, intensity and endurance performance in severely disabled MS patients. Forty-six progressive MS patients were randomized to receive 12 walking sessions of robot-assisted gait training (RAGT, n = 23) or conventional overground therapy (CT, n = 23). Ten healthy subjects were also studied. Blood samples were collected to determine lactate, pyruvate, and glutathione levels and lactate/pyruvate ratio pre&ndash, post rehabilitation. In vivo muscle metabolism and endurance walking capacity were assessed by resting muscle oxygen consumption (rmVO2) using near-infrared spectroscopy and by six-minute walking distance (6MWD), respectively. The levels of mitochondrial biomarkers and rmVO2, altered at baseline with respect to healthy subjects, improved after rehabilitation in the whole population. In the two groups, an enhanced response was observed after RAGT compared to CT for lactate (p = 0.012), glutathione (&lt, 0.001), lactate/pyruvate ratio (p = 0.08) and rmVO2 (p = 0.07). Metabolic biomarkers and 6MWD improvements were exclusively correlated with a training speed markedly below individual gait speed. In severely disabled MS patients, rehabilitation rebalanced altered serum metabolic and muscle parameters, with RAGT being more effective than CT. A determinable slow training speed was associated with better metabolic and functional recovery. Trial Registration: ClinicalTrials.gov NCT02421731.

Introduction Venous bed specificity could contribute to differential vulnerability to thrombus formation, and is potentially reflected in mRNA profiles. Materials and methods Microarray-based transcriptome analysis in wall and valve specimens from internal jugular (IJV) and saphenous (SV) veins collected during IJV surgical reconstruction in patients with impaired brain outflow. Multiplex antigenic assay in paired jugular and peripheral plasma samples. Results Most of the top differentially expressed transcripts have been previously associated with both vascular and neurological disorders. Large expression differences of HOX genes, organ patterning regulators, pinpointed the vein positional identity. The “complement and coagulation cascade” emerged among enriched pathways. In IJV, upregulation of genes for coagulation inhibitors (TFPI, PROS1), activated protein C pathway receptors (THBD, PROCR), fibrinolysis activators (PLAT, PLAUR), and downregulation of the fibrinolysis inhibitor (SERPINE1) and of contact/amplification pathway genes (F11, F12), would be compatible with a thromboprotective profile in respect to SV. Further, in SV valve the prothrombinase complex genes (F5, F2) were up-regulated and the VWF showed the highest expression. Differential expression of several VWF regulators (ABO, ST3GAL4, SCARA5, CLEC4M) was also observed. Among other differentially expressed hemostasis-related genes, heparanase (HPSE)/heparanase inhibitor (HPSE2) were up-/down-regulated in IJV, which might support procoagulant features and disease conditions. The jugular plasma levels of several proteins, encoded by differentially expressed genes, were lower and highly correlated with peripheral levels. Conclusions The IJV and SV rely on differential expression of many hemostasis and hemostasis-related genes to balance local hemostasis, potentially related to differences in vulnerability to thrombosis.

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Background and purpose The involvement of protein C (PC) pathway components in multiple sclerosis (MS) has scarcely been explored. The aim was to investigate their levels in relation to clinical and neurodegenerative magnetic resonance imaging (MRI) outcomes in patients. Methods In all, 138 MS patients and 42 healthy individuals were studied. PC, protein S (PS) and soluble endothelial protein C receptor (sEPCR) were evaluated by multiplex assays and enzyme-linked immunosorbent assay. Regression analyses between 3 T MRI outcomes and PC pathway components were performed. ancova was used to compare MRI volumes based on protein level quartiles. Partial correlation was assessed amongst levels of PC pathway components and hemostasis protein levels, including soluble thrombomodulin (sTM), heparin cofactor II (HCII), plasminogen activator inhibitor 1 (PAI-1) and factor XII (FXII). The variation of PC concentration across four time points was evaluated in 32 additional MS patients. Results There was an association between PC concentration, mainly reflecting the zymogen PC, and MRI measures for volumes of total gray matter (GM) (P = 0.003), thalamus (P = 0.007), cortex (P = 0.008), deep GM (P = 0.009) and whole brain (P = 0.026). Patients in the highest PC level quartile were characterized by the lowest GM volumes. Correlations of PC-HCII, PC-FXII and sEPCR-sTM values were detectable in MS patients, whilst PC-PS and PS-PAI-1 correlations were present in healthy individuals only. Conclusions Protein C plasma concentrations might be associated with neurodegenerative MRI outcomes in MS. Several differences in correlation amongst protein plasma levels suggest dysregulation of PC pathway components in MS patients. The stability of PC concentration over time supports a PC investigation in relation to GM atrophy in MS.

BACKGROUND Optimization of factor VIII (FVIII) infusion in hemophilia A would benefit from identification of FVIII pharmacokinetics (PK) determinants. The low-density lipoprotein receptor (LDLR) contains an FVIII-binding site and might influence FVIII clearance. Consistently, LDLR polymorphisms have been associated with FVIII levels. OBJECTIVE To investigate the relationships between individual FVIII PK and functional LDLR polymorphisms. PATIENTS/METHODS Thirty-three hemophilia A patients (FVIII coagulant activity [FVIII:C] ≤2 IU/dL) without inhibitors underwent 85 FVIII single-dose (21.4-51.8 IU/kg) PKs with different FVIII concentrates. Twenty patients underwent repeated PKs (2-6). FVIII C measured up to 72 hours was analyzed by two-compartment model. Parameters were evaluated in relation to F8 mutations, ABO blood-group and LDLR genotypes. RESULTS F8 mutation types were not associated with PK parameters. ABO and LDLR c.1773C/T polymorphism were associated with Alpha, Alpha HL, CLD2, K1-2, and K2-1 parameters, suggesting an influence on the FVIII initial distribution phase. Regression analysis showed an independent association of both ABO and LDLR c.1773C/T with PK parameters (Alpha, β-coefficient -0.311 vs 0.348; CLD2, β-coefficient -0.335 vs 0.318), giving rise to an additive effect in subjects stratified by combined phenotypes. Differently, the LDLR c.81C/T was associated with FVIII clearance and volume of distribution at steady state, which could be related to distinct effects of polymorphisms, potentially linked to LDLR intracellular distribution and FVIII binding behavior. CONCLUSIONS With the limitation of different FVIII concentrates and low number of patients, our data show plausible associations of LDLR polymorphisms with FVIII PK parameters, thus supporting their investigation as candidate functional determinants of FVIII PK.

In multiple sclerosis (MS), several adhesion molecules are involved within the central nervous system in inflammatory and neurodegenerative processes that are associated to progressive disability and increasing brain atrophy. The neural cell adhesion molecule (NCAM) has been suggested to participate in the reparative mechanisms and in the remyelination processes, key issues in MS pathology. We aimed at investigating plasma levels of the seldom investigated soluble (s)NCAM, and as comparison those of intercellular adhesion molecule-1 (sICAM-1) and vascular adhesion molecule-1 (sVCAM-1), and their association with clinical and MRI measures of lesion volumes and of global and regional atrophy. The cross-sectional study was conducted in 85 relapsing-remitting (RR)-MS, 53 progressive (P)-MS patients, and 42 healthy individuals (HI). Correlation of MRI measures with plasma levels of these adhesion molecules were not observed. In the MS and HI groups, sNCAM levels were significantly and positively associated with sVCAM-1 levels. Differently, the correlation between sICAM-1 and sVCAM-1 was observed only in MS patients. sNCAM and sVCAM-1 levels were higher in P-MS compared to HI (P = 0.05 and P = 0.028 respectively). The sVCAM-1 levels differed (P < 0.001) among DMTs groups and HI. The association of sNCAM plasma levels with MS disease, as well as differences in sVCAM-1 levels in patients receiving different DMTs, deserve further investigation.

Background Multiple sclerosis (MS) is an inflammatory, demyelinating and degenerative disorder of the central nervous system (CNS). Several observations support interactions between vascular and neurodegenerative mechanisms in multiple sclerosis (MS). To investigate the contribution of the extracranial venous compartment, we analysed expression profiles of internal jugular vein (IJV), which drains blood from CNS, and related plasma protein levels. Methods We studied a group of MS patients (n = 19), screened by echo-color Doppler and magnetic resonance venography, who underwent surgical reconstruction of IJV for chronic cerebrospinal venous insufficiency (CCSVI). Microarray-based transcriptome analysis was conducted on specimens of IJV wall from MS patients and from subjects undergoing carotid endarterectomy, as controls. Protein levels were determined by multiplex assay in: i) jugular and peripheral plasma from 17 MS/CCSVI patients; ii) peripheral plasma from 60 progressive MS patients, after repeated sampling and iii) healthy individuals. Results Of the differentially expressed genes (≥ 2 fold-change, multiple testing correction, P

Background: Chemokine ligands and co-stimulatory factors are involved in macrophage activation and differentiation processes that could contribute to multiple sclerosis (MS) pathogenesis. Objective: To investigate associations of C-C motif Ligand 18 (CCL18), C-C motif ligand 5 (CCL5) and soluble Cluster of Differentiation 86 (sCD86) with clinical and MRI measures in MS patients. Methods: Plasma levels of CCL18, CCL5 and sCD86 were evaluated in 138 MS patients (85 relapsing-remitting, RR-MS; 53 progressive, P-MS), and in 42 age- and sex-matched healthy individuals (HI). All subjects underwent standardized 3T MRI and clinical examinations. Multiple regression analysis of MRI outcomes as dependent variables was performed with age, gender, having P-MS, and plasma proteins as predictor variables. Results: Higher CCL18 plasma levels were found in P-MS (median = 51.5, IQR = 41.0–63.6 ng/mL) compared to RR-MS (median = 43.0, IQR = 29.1–55.0 ng/mL, p = 0.014) and to HI (median = 41.3, IQR = 30.9–54.1 ng/mL, p = 0.009). Disease-modifying treatments altered CCL5 (p = 0.036) and sCD86 (p < 0.001) levels. Higher CCL18 levels were associated with increased lateral ventricular volume (p = 0.006) and T2 lesion volume (LV) (p = 0.034), and decreased grey matter (p = 0.006), thalamic (p = 0.007) and cortical (p = 0.01) volumes. Conclusions: Our results provide evidence that higher CCL18 plasma levels are associated with more severe inflammatory and neurodegenerative brain MRI outcomes in MS.

Background and purpose The aim was to investigate the plasma levels of hemostasis components in multiple sclerosis (MS) and their association with clinical and magnetic resonance imaging (MRI) outcomes. Methods In all, 138 MS patients [85 with relapsing-remitting MS (RR-MS) and 53 with progressive MS (P-MS) with a mean age of 54 years; 72.5% female; median Expanded Disability Status Scale 3.5; mean disease duration 21 years] and 42 age- and sex-matched healthy individuals (HI) were studied. All subjects were examined with 3 T MRI and clinical examinations. Plasma levels of hemostasis factors [procoagulant, factor XII (FXII)] and inhibitors [tissue factor pathway inhibitor (TFPI), thrombomodulin, heparin cofactor II, a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) and plasminogen activator inhibitor 1 (PAI-1)] were evaluated by magnetic Luminex assays and enzyme-linked immunosorbent assay. Associations between hemostasis plasma levels and clinical and MRI outcomes were assessed. Results Lower ADAMTS13 levels were found in MS patients compared to HI (P = 0.008) and in MS patients presenting with cerebral microbleeds compared to those without (P = 0.034). Higher PAI-1 levels were found in MS patients compared to HI (P = 0.02). TFPI levels were higher in the P-MS subgroup compared to RR-MS patients (P = 0.011) and compared to HI (P = 0.002). No significant associations between hemostasis plasma levels and clinical or MRI outcomes were found. Conclusions Decreased ADAMTS13, particularly in MS patients with cerebral microbleeds, which deserves further investigation, and increased PAI-1 and TFPI levels were observed in MS patients, which deserves further investigation. No relationship between hemostasis plasma levels and measures of disease severity was detected.

Background Gait and mobility impairments affect the quality of life (QoL) of patients with progressive multiple sclerosis (MS). Robot-assisted gait training (RAGT) is an effective rehabilitative treatment but evidence of its superiority compared to other options is lacking. Furthermore, the response to rehabilitation is multidimensional, person-specific and possibly involves functional reorganization processes. The aims of this study are: (1) to test the effectiveness on gait speed, mobility, balance, fatigue and QoL of RAGT compared to conventional therapy (CT) in progressive MS and (2) to explore changes of clinical and circulating biomarkers of neural plasticity. Methods This will be a parallel-group, randomized controlled trial design with the assessor blinded to the group allocation of participants. Ninety-eight (49 per arm) progressive MS patients (EDSS scale 6–7) will be randomly assigned to receive twelve 2-h training sessions over a 4-week period (three sessions/week) of either: (1) RAGT intervention on a robotic-driven gait orthosis (Lokomat, Hocoma, Switzerland). The training parameters (torque of the knee and hip drives, treadmill speed, body weight support) are set during the first session and progressively adjusted during training progression or (2) individual conventional physiotherapy focusing on over-ground walking training performed with the habitual walking device. The same assessors will perform outcome measurements at four time points: baseline (before the first intervention session); intermediate (after six training sessions); end of treatment (after the completion of 12 sessions); and follow-up (after 3 months from the end of the training program). The primary outcome is gait speed, assessed by the Timed 25-Foot Walk Test. We will also assess walking endurance, balance, depression, fatigue and QoL as well as instrumental laboratory markers (muscle metabolism, cerebral venous hemodynamics, cortical activation) and circulating laboratory markers (rare circulating cell populations pro and anti-inflammatory cytokines/chemokines, growth factors, neurotrophic factors, coagulation factors, other plasma proteins suggested by transcriptomic analysis and metabolic parameters). Discussion The RAGT training is expected to improve mobility compared to the active control intervention in progressive MS. Unique to this study is the analysis of various potential markers of plasticity in relation with clinical outcomes. Trial registration ClinicalTrials.gov, identifier: NCT02421731. Registered on 19 January 2015 (retrospectively registered). Electronic supplementary material The online version of this article (doi:10.1186/s13063-017-1838-2) contains supplementary material, which is available to authorized users.

High levels of coagulation factor VIII (FVIII) have been associated with cardiovascular disease. Low-density lipoprotein receptor (LDLR) has been recently demonstrated to contribute to FVIII clearance from plasma. The aim of this study was to evaluate 3 single nucleotide polymorphisms in SMARCA4-LDLR gene locus (rs1122608, rs2228671, and rs688) and FVIII coagulant activity (FVIII:c) in subjects with (n = 692) or without (n = 291) angiographically confirmed coronary artery disease (CAD). High FVIII:c levels were an independent risk factor for CAD. The rs688 and rs2228671 genotypes were predictors of FVIII:c with T alleles associated with higher FVIII:c levels. The rs2228671T allele was associated also with reduced total and LDL-cholesterol levels. With respect to the risk of CAD, no association was found for rs2228671. Consistently with higher FVIII:c levels, the rs688T allele was associated with CAD, whereas, consistently with a favorable lipid profile, the rs1122608T allele was associated with a decreased CAD prevalence. After adjustment for classic cardiovascular risk factors, including plasma lipids, rs688 remained associated with CAD (OR for T carriers: 1.67 with 95% confidence interval, 1.10-2.54). Haplotype analysis confirmed such results. Our data suggest that polymorphisms at LDLR locus modulate FVIII:c levels and may be associated with CAD risk independently from plasma lipids.

Previous studies have established that factor VII gene (F7) polymorphisms (5′F7 and R353Q) contribute about one-third of factor VII (FVII) level variation in plasma. However, F7 genotyping in patients with cardiovascular disease has produced conflicting results. Population and expression studies were used to investigate the role of intron 7 (IVS7 ) polymorphisms, including repeat and sequence variations, in controlling activated FVII (FVIIa) and antigen (FVIIag) levels. Genotype–phenotype studies performed in 438 Italian subjects suggested a positive relation between the IVS7 repeat number and FVII levels. The lowest values were associated with theIVS7 + 7G allele. The screening of 52 patients with mild FVII deficiency showed an 8-fold increase in frequency (8%) of this allele, and among heterozygotes for identical mutations, lower FVII levels were observed in the IVS7 + 7G carriers. This frequent genetic component participates in the phenotypic heterogeneity of FVII deficiency. The evaluation of the individual contribution of polymorphisms was assisted by the expression of each IVS7variant, as a minigene, in eukaryotic cells. The novel quantitative analysis revealed that higher numbers of repeats were associated with higher mRNA expression levels and that the IVS7 + 7Gallele, previously defined as a functionally silent polymorphism, was responsible for the lowest relative mRNA expression. Taken together, these findings indicate that the IVS7 polymorphisms contribute to the plasmatic variance of FVII levels via differential efficiency of mRNA splicing. These studies provide further elements to understand the control of FVII levels, which could be of importance to ensure the hemostatic balance under pathologic conditions.

Several observations suggest the expansion of a distinct medial smooth muscle cell (SMC) subset in atherosclerosis and restenosis. We characterized the phenotypic features of SMC subsets in cultures derived from human carotid endarterectomy specimens. Specimens comprised an undiseased portion (thin intimal thickening with the underlying media) and a diseased portion (atherosclerotic plaque with the underlying media). From plaque tissues of the diseased portion, only macrophage-derived foam cells were retrieved. From medial tissues, two SMC phenotypes were isolated: large SMCs (flat with a monolayered growth pattern, from the undiseased portion) and small SMCs (fusiform and growing in multilayers, from the undiseased and diseased portions after co-culture with macrophage-derived foam cells). Small SMCs displayed higher proliferative and migratory activities and were less differentiated than large SMCs. Proteomic analysis showed that calmodulin was predominant in small SMCs. Co-culture of large SMCs with macrophage-derived foam cells induced a transition to the small phenotype with increased calmodulin expression. The calmodulin inhibitor W-7 decreased the proliferation of small SMCs and prevented the large to small phenotypic transition. In vivo, calmodulin was markedly expressed in SMCs of atherosclerotic plaques and was barely detectable in the media. Macrophage-derived foam cells promote selective migration from the media of atheroma-prone SMCs characterized by calmodulin overexpression. Further studies of small SMCs could be instrumental in understanding atherosclerosis pathogenesis and in planning therapeutic strategies.

Background: Different experimental observations suggest the expansion of a distinct medial smooth muscle cell (SMC) subset during atheromatous plaque establishment and restenosis. Objectives: We investigated the presence and features of such SMC subset in cell cultures derived from human carotid endarterectomy (CEA) specimens; moreover, we aimed at finding markers that could play a role in SMC behavior. Methods and Results: CEA specimens comprised a grossly undiseased (UP) and a diseased portion (DP). The UP contained a thin intimal thickening with the underlying media and the DP an atherosclerotic plaque with the underlying media. Cell cultures were initiated by tissue explantation. From plaque tissues only macrophage-derived foam cells were retrieved. From medial tissues 2 distinct SMC phenotypes were identified: 1) large SMCs, flat with a monolayered growth pattern, isolated from the UP media; 2) small SMCs, fusiform and growing in multilayers, isolated from both the UP and DP media only when cocultured with plaque-derived macrophages. Small SMCs displayed higher proliferative and migratory activities and a poor level of differentiation compared with large SMCs. Two-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry showed that calmodulin (CaM), a calcium-binding protein involved in cell-cycle regulation, was mainly expressed in small SMCs. This result was confirmed using a specific CaM antibody. Coculture of large SMCs with plaque-derived macrophages induced a switch to the small phenotype and was associated with increased CaM expression. Consistently, the specific CaM inhibitor W-7 hampered the large-to-small switch and predominantly inhibited the proliferation of small SMCs. In vivo, CaM was markedly expressed in atherosclerotic plaques whereas it was barely detectable in the media. Conclusions: Plaque-derived macrophages promote the selective migration of a distinct SMC subpopulation exhibiting features of an “atheroma-prone” phenotype. CaM is a marker of small SMCs in vitro and of plaque SMCs in vivo, and plays a role in SMC phenotypic modulation. Further studies on these SMC populations can be instrumental in understanding and influencing the evolution of atherosclerosis.

Molecular defects and polymorphic haplotypes of coagulation factor VII gene were studied in eight unrelated Italian subjects with factor VII deficiency, seven having the factor VII- variant, one the factor VIIR variant. An intron 7 mutation, which alters the consensus donor splice site sequence, was found in six subjects. The presence of the founder effect is suggested by their common geographical origin (a mountain area in the Lazio region) and by the identical polymorphic haplotype underlying the mutation. A different mutation, also located in the 5' monomer of the repeated intron 7 sequence, was found in the heterozygous condition in a subject from Northern Italy. New polymorphic alleles were detected in the repeated intron 7 region in subjects from Eastern Africa. Two missense mutations in codon 97 (Gly--Cys, Gly--Ser), the first found in the compound heterozygous condition with the frequent intron 7 mutation, suggest the presence of a hot spot mutation site in the second epidermal growth factor domain. Two neutral dimorphisms at codon 333Ser and 115His were detected, the last in linkage disequilibrium with the 353Arg/Gln polymorphism, and showing differences in frequency in the FVII deficient and control subjects.

Summary. Background: Combined vitamin K-dependent clotting factor (VKCF) deficiency type 2 (VKCFD2) is a rare bleeding disorder caused by mutated vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) gene. Methods and results: An Italian patient with moderate to severe bleeding tendency was genotyped, and found to be homozygous for the unique VKORC1 mutation (Arg98Trp) so far detected in VKCFD2. The activity levels of VKCFs were differentially reduced, and inversely related to the previously estimated affinity of procoagulant factor propeptides for the γ-carboxylase. The normal (factor IX) or reduced antigen levels (other VKCFs) produced a gradient in specific activities. Vitamin K supplementations resulted in reproducible, fast and sustained normalization of PT and APTT. At 24 h the activity/antigen ratios of VKCFs were close to normal, and activity levels were completely (factor VII and IX), virtually (prothrombin, factor X and protein C) or partially (protein S) restored. Thrombin generation assays showed a markedly shortened lag time. The time to peak observed at low tissue factor concentration, potentially mimicking the physiological trigger and able to highlight the effect of reduced protein S levels, was shorter than that in pooled normal plasma. At 72 h the thrombin generation times were normal, and the decrease in activity of procoagulant VKCFs was inversely related to their half-life in plasma. The improved coagulation phenotype permitted the uneventful clinical course after invasive diagnostic procedures. Conclusions: Modification of coagulation phenotypes in VKCFD2 after vitamin K supplementation was clinically beneficial, and provided valuable patterns of factor specific biosynthesis, half-life and decay.

Synovial Sarcoma (SS) is an aggressive neoplasm commonly affecting deep soft tissues of the extremities. In rare instances SS can arise in large veins of the lower extremities or trunk. We report the first case of intravascular synovial sarcoma (IVSS) occurring in a male patient. A biphasic tumor was diagnosed by histology and immunohistochemistry. Molecular analysis at RNA level confirmed the diagnosis demonstrating the chromosomal translocation t(X;18) (p11.2;q11.2) in the tumor. Although extremely rare, IVSS should be considered in the differential diagnosis of primary intravascular neoplasms and as a potential cause of deep vein thrombosis and thromboembolism.

Summary. Background: Nonsense mutations in coagulation factor (F) VII potentially cause a lethal hemorrhagic diathesis. Readthrough of nonsense mutations by aminoglycosides has been studied in a few human disease models with variable results. Objectives: We investigated the K316X and W364X FVII mutations, associated with intracranial hemorrhage, and their correction by aminoglycosides. The rare nonsense mutations in FVII represent favorite models to test this strategy, because even tiny increases in the amount of functional full-length protein in patients could ameliorate hemorrhagic phenotypes. Results: A FVII–green fluorescent protein (GFP) chimaera provided us with a fluorescent model of FVII expression in living cells. Appreciable fluorescence in cells transfected with nonsense FVII–GFP mutants was detected upon geneticin treatment, thus demonstrating suppression of premature translation termination. To investigate the rescue of FVII function, nonsense variants of the native FVII without GFP (p316X–FVII and p364X–FVII) were transfected and found to secrete low amounts of FVII (∼1% of Wt–FVII activity), thus suggesting a spontaneous stop codon readthrough. Geneticin treatment of cells resulted in a significant and dose-dependent increase of secreted FVII molecules (p316X–FVII, 24 ± 12 ng mL−1, 3.6 ± 0.8% of Wt–FVII activity; p364X–FVII, 26 ± 10 ng mL−1, 3.7±0.6%) characterized by reduced specific activity, thus indicating the synthesis of dysfunctional proteins. Similar results were observed with gentamicin, a commonly used aminoglycoside of potential interest for patient treatment. Conclusions: Our approach, extendable to other coagulation factors, represents an effective tool for a systematic study of the effects of aminoglycosides and neighboring sequences on nonsense codon readthrough. These results provide the rationale for a mutation-specific therapeutic approach in FVII deficiency.

Summary. Objective: To characterize the first type II protein S (PS) deficiency affecting the epidermal growth factor (EGF)4 domain, a calcium-binding module with a poorly defined functional role. Patients: The proband suffered from recurrent deep vein thrombosis and showed reduced PS anticoagulant activity (31%), and total, free PS antigen and C4bBP levels in the normal range. Results: Reverse transcription-polymerase chain reaction analysis showed the presence of the IVSg-2A/T splicing mutation that, by activating a cryptic splice site, causes the deletion of codons Ile203 and Asp204. Free PS, immunopurified from proband's plasma, showed an altered electrophoretic pattern in native condition or in the presence of Ca2+. The recombinant PS (rPS) mutant showed reduced anticoagulant (

The control of coagulation factor levels has a pivotal role in thrombophilia and known functional polymorphisms explain only a minor part of the coagulation factor level variance. Several studies provide evidence for i) interaction of polymorphisms and their combination with environmental factors; ii) molecular mechanisms mediating activation of coagulation proteins by environmental factors; iii) molecular mechanisms mediating transcriptional control of thrombophilia genes by environmental factors. However, the vast majority of molecular regulation by environmental factors is still unknown. Understanding of gene-gene and gene-environment interactions will require a detailed knowledge of regulatory regions of thrombophilia genes and of regulatory proteins.

Proteolytic cleavage of the peptide bond between Arg(152) and Ile(153) converts the procoagulant protein Factor VII (FVII) to an activated two-chain form (FVIIa). The formation of a salt bridge between Ile(153) and Asp(343) drives the conversion of FVIIa from being zymogen-like to the active form. In the present paper, we describe the novel FVII mutant V154G (Val(154)-->Gly mutation; residue 17 in the chymotrypsin numbering system), found in three FVII-deficient patients, which models a zymogen-like form of FVIIa. Recombinant V154G FVIIa, although normally cleaved, shows markedly reduced activity towards peptidyl substrate and undetectable activity towards macromolecular substrates. Susceptibility of Ile(153) to chemical modification, in either the presence or the absence of tissue factor (TF), suggests that the reduced V154G FVIIa activity is caused by impaired salt-bridge formation, thus resulting in a zymogen-like FVIIa form. The TF-mediated protection from chemical modification of V154A indicated that Gly(154) is responsible for this peculiar feature, and suggests that this region, proximal to the heavy chain N-terminus, is directly involved in the conversion of FVII into FVIIa. V154G FVII was exploited to study the FVII-TF interaction, together with three additional FVII variants that were expressed to serve as models for different FVII forms. The comparison of binding affinities of full-length TF after relipidation in L-alpha-phosphatidylcholine for the zymogen FVII (Arg(152)-->Gln, K (d)=1.04+/-0.27 nM), inactive FVIIa (Ser(344)-->Ala, K (d)=0.27+/-0.06 nM) and a zymogen-like FVIIa (V154G, K (d)=1.15+/-0.16 nM) supports the hypothesis that preferential binding of TF to active FVIIa is insufficient to drive the 10(5)-fold enhancement of FVIIa activity. In addition, the inability of V154G FVIIa to accommodate an inhibitor in the active site, indicating an improperly shaped specificity pocket, would explain the low activity of the zymogen-like form of FVIIa, which is predominant in the absence of TF.

Abstract —The present analysis was undertaken to study the effect of oral contraceptive (OC) use on activated factor VII (FVIIa) in subjects characterized by FVII genotypes, with the further aim of evaluating the role of lipids in this pharmacological interaction. In OC users (n=42) and nonusers (n=130) of comparable age, we examined the FVII phenotypic variables (FVII coagulant activity [FVIIc], FVII antigen, and FVIIa), FVII genotypes (the 353R/Q and 5′F7 polymorphisms analyzed in combination; alleles M1 / M2 and A1 / A2 , respectively), and a number of lipid and lipoprotein parameters: serum concentrations of total cholesterol (chol), low density lipoprotein and high density lipoprotein-chol, triglycerides, phospholipids (PhLs), apolipoprotein A1, and lipoprotein(a). PhLs, triglycerides, apolipoprotein A1, chol, FVII antigen, FVIIc, and high density lipoprotein-chol levels were shown to be statistically higher in users than nonusers. FVII levels, particularly those of FVIIa and FVIIc, were much higher in homozygotes for the A1 and M1 alleles ( A11 M11 ), especially in OC users. A strong association was found between PhL and FVIIa: in the multiple regression analysis, women taking OCs who had elevated PhL concentrations also had very high levels of FVIIa, but only if their genotype was A11 M11 . These results indicate that the increased FVII levels in OC users depend on the FVII genotype and that high PhL concentrations predict very high levels of FVIIa and FVIIc.

Abstract A genetic variation in the 3′-untranslated region of the prothrombin mRNA (20210 G/A) has recently been reported to be associated with elevated plasma prothrombin levels and with an increased incidence of venous thrombosis. We determined the frequency of this mutation, the detection of which was improved by allele-specific amplification of exon 14 and by denaturing gradients (denaturing gradient gel electrophoresis), in cohorts of patients affected by venous thrombosis (n=132) or by coronary or cerebrovascular diseases (n=195) and in normal subjects from various populations. An overlapping frequency of the heterozygous genotype (4%) was found in normal subjects from Italy and Cyprus, and no carrier was detected in 40 subjects of Indian or Somali origin. The 20210 GA heterozygous genotype was not increased in frequency in patients with arterial disease. In contrast, the GA genotype was associated ( P =.007) with venous thrombosis both in simple heterozygotes (16%) with a family history of thrombosis as well as in double heterozygotes (14%) for other known thrombophilic defects. A synergic interaction between the prothrombin 20210 GA genotype and the factor V Leiden mutation, both potentially affecting the prothrombinase complex, was suggested by the early onset of thrombosis (median age 22 years) in doubly heterozygous patients. The association of the 20210 A allele with higher prothrombin levels was confirmed in the Italian population. However, the prothrombin assay does not allow an efficient preselection of patients for the DNA analysis.

Abstract To assess whether certain abnormalities of the sulfated amino acid metabolism are associated with the occurrence of thromboembolic events in patients with inherited thrombophilic conditions, the levels of homocyst(e)ine, before or after methionine load, and the presence of the Ala 223 Val substitution in the 5,10-methylenetetrahydrofolate reductase (MTHFR) were evaluated in 119 subjects with a congenital single thrombophilic condition (type I deficiency of antithrombin n=10, protein C n=24, protein S n=16; activated protein C resistance due to factor V Leiden mutation n=69). Sixty-three subjects had experienced at least one documented thrombotic event, while the remaining 56 subjects were still free from any thrombotic symptom. Our results show that (1) high homocyst(e)ine levels, either in fasting condition or after methionine load, were not more frequent in subjects with inherited thrombophilic alterations (14.4%) than in normal control subjects (10% by definition) and (2) the frequency of hyperhomocyst(e)inemia was similar in thrombophilic subjects, who already have (14.3%) or have not (14.6%) experienced thrombotic events. As regards the MTHFR mutation, the homozygous condition was present in 23.2% of the thrombophilic patients versus 17.5% in the control subjects, a nonsignificant difference. The mutation was slightly more frequent in those thrombophilic subjects who had suffered a thrombotic episode (25.5%) versus those with no thrombosis (20.8%), with odds ratios of 1.61 (confidence interval (CI)=0.58-4.52) and 1.24 (CI=0.42-3.43), respectively. These differences were also nonsignificant. It is concluded that in subjects with inherited thrombophilias, a condition of hyperhomocyst(e)inemia “per se” is not a factor increasing the risk of thrombosis. The risk enhancement conferred by the MTHFR mutation, if any, seems to be slight or limited, and its significance could be ascertained only in a large multicenter trial.

The 3' portion of the coagulation factor VII gene, containing the activation and serine protease domains, was investigated in four subjects with factor VII deficiency by temperature gradient gel electrophoresis and sequencing of polymerase chain reaction (PCR) products. Molecules displaying an altered melting behaviour were detected in three subjects, and direct sequencing showed two mutations. A G-to-T transversion causing a missense mutation, Cys-310 to Phe, suppresses a disulphide bond conserved in the catalytic domain of all serine proteases. This mutation, which in the homozygous form causes a severe reduction in protease activity (4%), was found in two patients from different Italian regions. A G-to-A transition, which gives rise to a missense mutation, Arg-304 to Gln, and is associated with the factor VII padua variant, was found in the heterozygous form in a subject also affected by von Willebrand disease. Two polymorphic alleles, which differ in one repeat monomer element, were precisely mapped in a region spanning the exon-intron 7 border of the factor VII gene and studied in families with factor VII or X deficiency.

An abnormal von Willebrand factor (vWF) gene restriction pattern has been found in a patient with von Willebrand disease. Because this gene alteration is not present in his parents or in 50 normal and 25 affected subjects, and the restriction fragment length polymorphism haplotypes are inherited normally in the patient's family, we suggest that a de novo mutation is present. Bands with reduced intensity and additional fragments, observed in several restriction digests, hybridize with noncontiguous copy DNA (cDNA) portions, thus indicating the presence of a heterozygous gene deletion. The deletion removes a genomic region containing at least codons 1147 through 1854 and corresponding to the D3-A3 homologous protein domains. The extent of the vWF pseudogene on chromosome 22 is roughly similar to that of the deleted area. However, the pseudogenic nature of the deletion is excluded by the mapping of bands with reduced intensity in the patient to the true vWF gene. The vWF antigen levels are one fourth of normal and ristocetin cofactor activity is severely impaired. The reduction of high molecular weight multimers in plasma and platelets and the altered triplet morphology are compatible with the presence of a dominant variant of type II von Willebrand disease.

The presence and inheritance of restriction fragment length polymorphisms (RFLPs) and gene lesions in the coagulation factor VIII gene were investigated in 15 hemophilia families. An abnormal HindIII 2.6-kb band, previously detected in a severe hemophiliac, was observed in a not severely affected patient and also in the normal gene of a woman carrying a hemophilic gene in which the lesions was found. The TaqI site in exon 24 of this defective gene was removed by a C to T transition causing an amino acid change (Arg----Gln). Very low amounts of factor VIII activity and antigen were detected in the severely affected grandson. The presence of the HindIII 2.6-kb fragment in both normal and pathological genes indicates that a factor VIII RFLP without functional meaning was found. Its frequency, determined in 60 chromosomes, is 0.18. Double digestions enabled us to map the polymorphic site 3' to the exon 19.

The von Willebrand factor pseudogene, previously mapped to chromosome 22, was sublocalized by in situ hybridization using as probe a von Willebrand factor cDNA fragment completely contained in the pseudogenic region. Chromosome spreads were from a patient carrying a unique balanced de novo translocation 46,X,t(X;22)(pter;q11.21). Silver grain analysis indicated that the human von Willebrand factor pseudogene is located on 22q,11,22-q11,23, a region relevant for several somatic and constitutional chromosomal alterations.

DNA from 20 Italian haemophilia B patients was analysed by the Southern blotting technique and hybridisation to a factor IX cDNA probe. A large deletion of factor IX gene was detected in one patient with antibodies to the infused factor; the EcoRI pattern of the other 19 subjects examined was normal.

To investigate the sporadic occurrence of hemophilia A and to estimate the sex ratio of mutation rates directly, 17 families with isolated cases of the disorder were studied by RFLP analysis and by clotting assays. Three RFLPs, one intragenic and two with close linkage to hemophilia A, were used. In eight families the RFLP study excluded the carrier status of the maternal grandmothers. Since hemostatic studies showed that the eight mothers of these propositi were hemophilia carriers, the origin of the newly mutated genes was inferred from the RFLP patterns: six hemophilic genes derived from the normal maternal grandfathers and two, from maternal grandmothers. The data indicate a higher mutation rate in males than in females, as previously suggested by segregation analysis and coagulation studies. However the sex ratio indicated by the RFLP analysis is lower than previously reported and could explain previous conflicting estimates.

The form of alpha-thalassaemia of the Po river delta presents haematological and globin biosynthetic characteristics similar to alpha-thalassaemia-1 but never gives rise to HB H disease nor to hydrops foetalis. In alpha-thalassaemic subjects originally from this region globin mRNA translation and alpha-globin gene arrangement have been investigated. The data obtained indicate that alpha-globin synthesis and reticulocyte alpha-globin mRNA are reduced by one fourth; in addition, since the defect in alpha-globin synthesis does not change with cell ageing, a possible instability of alpha-globin mRNA is excluded. Restriction enzyme analysis of the DNA shows a normal alpha-globin gene organization. This form of alpha-thalassaemia is therefore of the non-deletion type; its molecular lesion is either at the level of alpha-globin mRNA transcription or processing. The fact that this, as well as other forms of non-deletion alpha-thalassaemia, have a phenotypic expression similar to alpha-thalassaemia 1 is discussed.

Six beta(+)-thalassaemic patients from the Po river delta region have been studied. Using synthetic oligonucleotides as specific hybridisation probes, the beta(+) IVS I mutation (G----A at position 108) was demonstrated. This lesion and the enzyme polymorphism pattern in the subjects examined are the same as have been described for other Mediterranean beta(+)-thalassaemias. Antenatal diagnosis through DNA analysis of beta(+)-thalassaemia is therefore possible. The production of beta globin in a beta(+), homozygote and in a beta (+), beta(0) 39 (nonsense mutation at codon 39) double heterozygote is approximately 20% and 10% respectively of total non-alpha globin synthesis. Despite some overlapping of the results, similar beta globin synthesis levels have been obtained in 43 beta(+)-thalassaemia patients. This suggests that in the Po river delta region the most common thalassaemic genes are beta(0) 39 and beta(+) IVS I.

We have studied the expression of the c-myc and c-abl oncogenes in two human leukemic K562 cell lines which do express hemoglobin genes retaining a differential rate of cell proliferation. Our data indicate that in hemin-induced K562(S) cells the expression of c-abl oncogene decreases and appears to be related to a decrease in the proliferation capacity rather than to the activation of differentiated functions. The K562(hC) cell line, which produces large amounts of Hb Gower 1 retaining an efficient rate of cell proliferation, expresses indeed the c-abl oncogene at high level.