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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.

Rehabilitative exercise outcomes and plasma concentrations of soluble adhesion molecules (sEndoglin, sE-Selectin, sL-Selectin, sICAM-1, sNCAM, sNCAM-1, sVCAM-1, sPECAM-1, sVAP-1) were evaluated in 60 severely disabled progressive multiple sclerosis (MS) patients at 4-time points. Changes of sE-Selectin, sL-Selectin, and sPECAM-1 concentrations were observed over time, and their variations were significantly correlated with rehabilitative outcome variations. Baseline sVAP-1 concentrations were able to predict functional mobility recovery. Our data suggest that the evaluation of adhesion molecules in plasma provides useful information to interpret rehabilitative exercise processes and to identify potential predictors of the rehabilitation-induced changes in mobility outcomes in MS patients.

Background Transcranial direct current stimulation (tDCS) is used for therapeutic purpose in severely brain-injured patients. The relationship between the recovery after tDCS and potential biomarkers in plasma has been limitedly investigated in patients with minimal conscious state (MCS). Objective To investigate soluble neuronal adhesion molecule (sNCAM) plasma levels in relation to tDCS and recovery processes in MCS. Methods sNCAM was measured in plasma before (T−1,T0), during (T1) and after (T2, T3) tDCS sessions in eight patients with a post traumatic etiology and at least one year of chronic state. Results While sNCAM levels were highly correlated overtime, no significant difference was observed in relation to tDCS. An inverse relation was observed between sNCAM levels at baseline and the tDCS long-lasting effects (T−1, r = −0.852, p = 0.007; T0, r = −0.787, p = 0.020). Conclusions This exploratory research suggests the sNCAM levels, potentially associated with tDCS outcomes, as a candidate biomarker of neurobiological after-effects in MCS patients.

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.

Background Increasing evidence for contribution of hemostasis components in multiple sclerosis (MS) has been reported. Hemostasis protein inhibitors display key regulatory roles, extending to regulation of innate immune response and inflammation, and promotion of blood–brain barrier integrity. Whereas the effects on hemostasis of exercise and rehabilitation strategies have been extensively investigated, relationships between MS rehabilitation strategies and hemostasis have not been previously reported. Objectives To investigate in MS patients the association between outcomes of rehabilitative exercise and plasma levels of selected hemostasis inhibitors. Methods Sixty-one severely disabled progressive-MS (P-MS) patients were randomized in the RAGTIME trial to receive 12 walking session of robot-assisted gait training (RAGT) or conventional overground therapy (CT). Outcome parameters were: timed 25-foot walk test (T25FWT) speed, 6-minute walking test (6MWT), Berg Balance Scale (BBS), and MS impact scale-29 (MSIS-29). Plasma levels of coagulation inhibitors protein S (PS), soluble thrombomodulin (sTM), and tissue factor pathway inhibitor (TFPI) were assayed by multiplex assay and ELISA at 4-time points: baseline (T0), intermediate (T1), end of rehabilitation (T2), 3-month follow-up (T3). Descriptive analysis, trend analysis, Spearman's rank and Pearson's correlations, and multiple regression models were used. Results Rehabilitative exercises moderately modified plasma protein concentrations. A significant trend to increase was observed for PS (p=0.015) and TFPI (p=0.047) in the whole population, and for PS (p=0.011) in the CT group. Correlation between TFPI and sTM levels was detectable at all time points in the whole P-MS patients and in RAGT group. The correlation between TFPI and PS, present at T0, was lost during the rehabilitation, and recovered at T3 in the whole population and CT group. During rehabilitation, positive variations of TFPI were inversely related with changes in 6MWT in the whole population (r=-0.309, p=0.021), and in the RAGT group (r=-0.51, p=0.004). In all P-MS, PS T0 levels were associated (r=0.379, p=0.004) with increased gait speed, which in the RAGT group was associated both with PS T0 (r=0.378, p=0.040), and sTM T0 (r=0.453, p=0.012). Accordingly, in the regression model including age, sex and EDSS and the stepwise enter of PS T0, higher PS T0 levels predicted increased gait speed in all P-MS (F=3.4, p=0.016) The regression model in the RAGT group indicated that higher PS and sTM T0 levels were both predictors of increased gait speed (F=5.7, p=0.001). Conclusions Plasma levels of coagulation inhibitors were related to variations of outcome measurements after high-intensity walking rehabilitation programs. Patients with decreased TFPI levels from T0 to T2 displayed the most significant functional recovery following rehabilitation, and particularly after RAGT. Higher baseline total PS levels were associated with favorable outcomes of rehabilitation therapies in MS. These novel findings, which suggest that plasma levels of hemostasis inhibitors might have implication for rehabilitative therapy options in MS, warrant further investigation.

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.

Not available

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.

How genetic variations mediate normal and abnormal biological function is a major issue in biology and medicine. The enormous number of genomic sequences, and their frequent and rare variations identified in humans, require efficient approaches aimed at dissecting functional correlates. In this review we will focus on the importance of the assessment of well-defined intermediate phenotypes, on the set up of transcriptomic approaches in diseased cells and on the modulation of expression by sequence variations modulating mRNA splicing or influencing protein multimerization. These information provide the molecular bases of associations discovered through genomic approaches, and might open new avenues toward the design of novel and specific diagnostic, prophylactic or therapeutic interventions. Taking into account our previous and current experimental activities we shall focus on a few examples and open issues in cardiovascular disorders, the main clinical topic of this short review.

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.

In patients with congenital FVII deficiency, bleeding manifestations and clinical presentation vary widely, ranging from asymptomatic subjects to patients with haemorrhages that may cause important handicaps. Owing to menorrhagia, which occurs in about two-thirds of women of fertile age, bleeding is more frequent in women than in men. Gum bleeding and easy bruising are also more frequent in females. FVII:C levels are not a good predictor of bleeding tendency as there is a wide overlap between bleeders and asymptomatic patients. We propose a three-grade system of classification based on clinical considerations. Therapy for congenital FVIII bleeding is discussed, with the advantages and disadvantages of each treatment, and the suggested single dose given.

Summary. We investigated the role of frequent fibrinogen polymorphisms in venous thromboembolic disease in conjunction with inherited thrombophilia. Two hundred unrelated subjects, all carriers of the factor V R506Q mutation (FV Leiden), were genotyped at the fibrinogen gene cluster. Among these subjects, 100 had experienced previous venous thromboembolism (VTE) and 100 were still asymptomatic for VTE. Significant differences were observed between the groups for the BclI polymorphism (P = 0·004). Scanning, by sequencing the DNA regions flanking the BclI marker, revealed new polymorphisms, a C to T transition and a G to T transversion at 1520 and 3369 base pairs 3′ to the β gene stop codon respectively. These markers showed less association with the clinical phenotype than BclI itself. A combined genotype including 10 markers was more frequent among the asymptomatic subjects (17%) than among patients (3%), and was associated with a reduction in fibrinogen antigen level (2·42 ± 0·35 vs 2·69 ± 0·41 g/l, P = 0·028) among the asymptomatic subjects. Our data suggest that, in the presence of inherited thrombophilia, frequent fibrinogen polymorphisms may interact to modulate the risk of venous thromboembolism.

SummaryWe characterized a symptomatic CRMred factor X (FX) deficiency produced by the Glu19Ala mutation in the γ-carboxyglutamic-rich domain. FX activity levels in plasma were markedly reduced in prothrombin time assays (< 1-5%), whereas in activated partial thromboplastin assays (16%) and in RVV assays (17%) the reduction in activity mirrored that in antigen levels (17%). Activation of recombinant 19Ala-FX by factor IXa/factor VIIIa or RVV, and the activity in thrombin generation assays, were comparable to those of wild-type FX. Differently, complete activation of recombinant 19AlaFX required a factor VIIa/TF concentration 30-fold higher than that of wild-type FX. The recombinant FVIIa significantly reduced PT values in 19Ala-FX reconstituted plasma, thus suggesting an alternative approach for treatment of FX deficiencies characterized by defective FX activation.The study of this FX deficiency provides an “in vivo” and “in vitro” model for the investigation of Gla domain interactions.

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.

A total of 122 subjects, referred after presurgery screening or checkup for prolonged prothrombin time, were characterized for the presence of coagulation factor VII deficiency. Fourteen subjects carried a partial and asymptomatic deficiency, and in half of them dysfunctional molecules were detected in plasma. In nine subjects we found five missense mutations differing from those previously found in factor VII deficient patients. The others were homozygous for a common polymorphism (R353Q) that affects factor VII levels. A new codon dimorphism (A330) was also found in exon 8. Four mutations (R223W, M298I, R304Q, and R353Q) located at FVII-specific residues point out protein regions that are important for coagulation factor evolution, and two mutations (G342E and E265K) affect generic or partially generic residues. The newly reported mutations were combined with those we previously found, totalling 17 independent mutations responsible for FVII deficiency in 27 Italian pedigrees. We observed several similarities with the mutation pattern determined in factor IX, which include a high percentage of transitions at CpG doublets, the presence of hot spot sites affected by multiple substitutions, and of several topologically equivalent mutations. © 1996 Wiley-Liss, Inc.

Summary. The presence of gene lesions in coagulation factor X (FX, Stuart factor) was investigated in asymptomatic subjects with FX deficiency characterized by the presence of dysfunctional molecules in plasma, as demonstrated by the discrepancy between clotting activity and antigen level. A missense mutation (Ser334Pro) in the catalytic domain was found in three unrelated families in both the homozygous and the heterozygous conditions, and also in the compound heterozygous form with the substitution of Lys for 102 Glu. None of the mutations was detected in 40 unrelated subjects from the same geographic area. The Ser334Pro mutation affects a serine protease region characterized by extensive variation in the coagulation factors but conserved in mammalian factor X molecules. The Glul02Lys mutation affects a residue of the second EGF-like module also conserved in protein C. Both mutated residues are surface-exposed and found in protein regions suggested to be involved in macromolecular interactions which are impaired in the dysfunctional molecules.

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.

von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, is very heterogeneous and has been classified into several subtypes. Missense mutations have been found to be responsible for the dominant type II vWD, characterized by qualitative abnormalities affecting von Willebrand factor (vWF) function. The breakpoints of a heterozygous vWF gene deletion (31 Kb), occurring 'de novo' in a patient with a variant of type II vWD, were localized to introns 25 and 34 and sequenced. An Alu repeat in intron 25 was interrupted between the transcriptional boxes A and B. The new junction present in the abnormal von Willebrand factor mRNA was sequenced after reverse transcription of platelet RNA. The codon 1104 (Cys) is followed in frame by the mutated codon 1926 (Cys to Arg), thus removing the complete A domains, found in a wide variety of genes and characterized by independent assembly 'in vitro'. We propose that the abnormal vWF, which carries intact protein domains responsible for vWF dimer and multimer formation, makes ineffective interactions with the normal molecules in the biosynthetic process, causing the dominant type II phenotype through a novel mechanism.

Introduction Protein S (PS) is a vitamin K-dependent plasma glycoprotein with a key role in the control of coagulation pathway on phospholipid membranes. We compared anticoagulant and membrane binding properties of PS altered by natural mutations (N217S, DelI203D204) affecting the epidermal growth factor like-domain 4 (EGF4) and causing PS deficiency. Materials and methods Binding of recombinant, immunopurified PS (rPS) to several conformation-specific antibodies, to C4BP and to phospholipid liposomes was investigated by ELISA. PS binding to cells was analysed by flow cytometry. PS inhibitory activities were studied in plasma and purified systems. Results and conclusions Conformational changes produced by mutations were revealed by mapping with calcium-dependent antibodies. The immunopurified recombinant mutants (rPS) showed at 200-800nM concentration reduced inhibition of coagulation (rPS217S, 10.2-17.3%; rPSDelI203D204, 5.8-8.9% of rPSwt) in FXa 1-stage clotting assay with APC. In thrombin generation assays the inhibition of ETP was reduced to 51.6% (rPS217S) and 24.1% (rPSDelI203D204) of rPSwt. A slightly shortened lag time (minutes) was also observed (rPS217S, 2.58; rPSDelI203D204, 2.33; rPSwt, 3.17; PS deficient plasma, 2.17). In flow cytometry analysis both mutants efficiently bound apoptotic cells in adhesion or in suspension. The affinity for phosphatidylserine-rich vesicles (apparent Kd: rPSwt 27.7 ± 1.6 nM, rPS217S 146.0 ± 16.1 nM and rPSDelI203D204 234.1 ± 28.1 nM) was substantially increased by membrane oxidation (10.9 ± 0.6, 38.2 ± 3.5 and 81.4 ± 6.0 nM), which resulted in a virtually normal binding capacity of mutants at physiological PS concentration. These properties help to define the molecular bases of PS deficiency, and provide further elements for PS-mediated bridging of coagulation and inflammation.

Introduction Factor X (FX) is a serine-protease playing a crucial role in the blood coagulation pathway and triggering intracellular signalling in a variety of cells via protease-activated receptors (PARs). By exploiting naturally occurring variants (V342A and G381D, catalytic domain; E19A, γ-carboxyglutamic acid (GLA)-rich domain), we investigated the relationship between the pro-coagulant activity and the signal transduction capacity of FX. Materials and methods Recombinant FX (rFX) variants were expressed in Human Embryonic Kidney cells and purified by immunoaffinity chromatography. Activated rFX (rFXa) variants were characterized for pro-coagulant, amidolytic and thrombin generation activity. rFXa signalling was assessed through evaluation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in C2C12 myoblasts. Results and conclusions rFX variants showed reduced (rFX-342A, 29%; rFX-19A, 12%) or not detectable (rFX-381D) amidolytic activity. Thrombin generation activity in a plasma system was also decreased either upon activation by Russell's viper venom (rFX-342A, 38%; rFX-19A, 7%; rFX-381D, not detectable) or by the extrinsic pathway (rFX-342A, 36%; rFX-19A, rFX-381D, not detectable). The rFXa-381D mutant displayed little or no enzymatic activity, and did not induce any appreciable signal transduction capacity. The rFXa-342A mutant induced a dose-dependent signalling with a 50% reduced signalling capacity. At the highest concentration (174 nM), signalling progressed with a time course similar to that of rFXa-wt. Zymogen rFX-19A showed defective and incomplete activation resulting in strongly reduced enzymatic activity and signalling. Taken together our data are consistent with a close correlation between pro-coagulant activity and intracellular signalling capacity.

Summary The homologous pseudogenic and genic regions of von Willebrand factor (vWF) were studied in DNA from a patient with homozygous deletion of vWF genes and compared with a normal control. This analysis indicates informative restriction patterns for the investigation of restriction fragment length polymorphisms (RFLPs) and gene lesions, and for molecular cloning. A useful new genic Xbal RFLP was found and characterized. A large BgIII fragment of the pseudogenic region was cloned and mapped, and single sequences (9 kb) were used as probes. Corresponding genic and pseudogenic fragments, which contain exons 23–28, and specific restriction patterns were identified, including a new polymorphic TaqI site that was mapped in the gene. A cloned fragment contains the 5’boundary of the pseudogene and recognizes an additional and unknown homologous sequence in the genome. The chromosomal localization of the vWF pseudogene and of the breakpoint cluster region (BCR) gene were compared by ‘in situ’ hybridization: overlapping patterns were detected. The cloning, characterization and mapping of the pseudogenic region improves the analysis of this portion of chromosome 22 affected by several somatic and constitutional alterations, and also of the corresponding genic region on chromosome 12.

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.

Summary Three TaqI restriction fragment length polymorphisms (RFLP) detected by the central portion of von Willebrand factor cDNA, which recognizes the true gene and in addition pseudogenic sequences, were characterized and mapped. Small cDNA fragments which hybridized with DNA from families with von Willebrand disease were used. Two of the RFLP, recognized by 1.7 and 0.45 kb cDNA fragments, are not in linkage either with von Willebrand disease or with RFLP located in the von Willebrand factor (vWF) gene, which indicates their pseudogenic location. These markers located in 22q11, near to the bcr gene, provide new tools for the study of several somatic and constitutional alterations affecting this chromosomal region. The third RFLP is recognized by a cDNA fragment corresponding to the N-terminal portion of mature vWF and is localized in the true gene. Since significant linkage disequilibrium with other informative RFLP is not present, this marker contributes to the definition of family haplotypes associated with von Willebrand disease.