Abstract Background Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. The disruption of the hepatic interaction between Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) and Low-Density Lipoprotein Receptor (LDLR) downregulates blood cholesterol levels and reduces cardiovascular events. Recent data suggest that other members of the LDLR superfamily may be targets of PCSK9. Purpose The aim of this work is to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target, and to study the role of PCSK9 and LRP5 in foam cell formation and hence, in the mechanism of lipid accumulation and atherosclerotic plaque formation. Methods Intracellular protein and lipid localization, cholesteryl esters (CE) accumulation; quantification of structural and inflammatory proteins expression and immunoprecipitation analyses were performed in primary cultures of human inflammatory cells (monocytes and macrophages) silenced for LRP5 or PCSK9 and challenged with modified LDLs. Results We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages have less intracellular CE accumulation. In LDL treated macrophages internalization of LRP5-bound LDL starts after 30 minutes of incubation and lasts up to 24hours. The SREBP-2 promoter is not involved in LRP5 regulation but it does regulate macrophage PCSK9 expression. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally we studied the role of PCSK9 and LRP5 in the inflammatory response by TLR4/NFkB signaling pathway. We show decreased TLR4 protein expression levels and decreased nuclear translocation of NFκB in PCSK9 silenced-inflammatory cells after lipid loading indicating a downregulation of the proinflammatory pathway TLR4/NFκB. Increased gene expression is observed in TNF-α and IL1β after lipid-loading that is abolished in PCSK9-silenced macrophages. Furthermore release of the proinflammatory cytokines TNF-α and IL1β is decreased in PCSK9-silenced macrophages. LRP5 protein expression is increased in lipid-loaded macrophages independent of the presence or absence of PCSK9. Conclusions These results demonstrate that, in human macrophages, LRP5 is internalized with lipids. Furthermore, PCSK9 and LRP5 can form a complex in the cytoplasm of lipid-loaded macrophages opening the possibility that PCSK9 induces lysosomal LRP5 degradation in a similar manner than it does with LDLR. Finally we also show that PCSK9 gene interference decreases inflammation and supports a role for PCSK9 as an inflammatory mediator in atherosclerosis. Acknowledgement/Funding CN16/11/00411-LB; TERCEL RD16/0011/018-LB; FIS2016-02014 -MBP; SEC2015 to MBP

Background 2-GlycoproteinI (2-GPI), also designated apolipoproteinH, is a 50-kDa protein that circulates in blood at high concentrations, playing important roles in autoimmune diseases, hemostasis, atherogenesis, and angiogenesis, as well as in host defense against bacteria and in protein/cellular waste removal. Plasma 2-GPI levels have a significant genetic component (heritability of similar to 80%). Objectives To present the results of a genome-wide association study for plasma 2-GPI levels in a set of extended pedigrees from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. Patients/Methods A total of 306 individuals for whom 2-GPI plasma measurements were available were typed for 307984 single-nucleotide polymorphisms (SNPs) with the Infinium 317k Beadchip (Illumina). Association with the 2-GPI phenotype was investigated through variance component analysis, and the most significant results were followed up for association with coronary artery disease (CAD) in an independent insilico analysis involving 5765 CAD cases from the PROCARDIS Project and 7264 controls from the PROCARDIS Project and the Wellcome Trust Case Control Consortium (WTCCC) collection. Results After correction for multiple testing, three SNPs located in/around two genes (ELF5 and SCUBE2) reached genome-wide significance. Moreover, an SNP in the APOH gene showed suggestive association with the 2-GPI phenotype. Some of the identified genes are plausible biological candidates, as they are actually or potentially involved in inflammatory processes. Conclusions Our results represent a first step towards identifying common variants reflecting the genetic architecture influencing plasma 2-GPI levels, and warrant further validation by functional experiments, as the functions of some of the discovered loci are still unknown.

Low-molecular-weight heparins (LMWHs) are antithrombotic drugs that differ on biochemical and pharmacological properties. Objective: This study was conducted to compare the pharmacodynamic time-course of two LMWHs, bemiparin and enoxaparin, at high prophylactic doses. Methods: This was an open, randomized, single-blind, cross-over study to compare the pharmacodynamic time-course, safety and tolerability of two LMWHs, bemiparin 3500 IU and enoxaparin 4000 IU at subcutaneous single doses in 12 healthy male volunteers. Anti-Xa activity (main biomarker of heparin activity), anti-IIa activity, total and free tissue factor pathway inhibitor (TFPI), activated partial thromboplastin time (APTT), thrombin time (TT) and thromboplastin-thrombomodulin mediated time (Tp-TmT) were investigated. Results: Bemiparin 3500 IU achieved more anti-Xa activity than enoxaparin 4000 1 U, measured by the area under the curve (geometric mean AUC(0)(1)) (bemiparin 3.69 vs. enoxaparin 3.33 IU h/ml; p < 0.001). Maximum anti-Xa activity was reached at 3 hours and there were anti-Xa measurable levels up to 16 h after subcutaneous administration. Anti-Xa activity half-life was 5.44 hours for bemiparin and 4.71 hours for enoxaparin. Anti-IIa activity was above the limit of quantification (0.05 IU/ml) in only 2 volunteers after bemiparin and in 8 after enoxaparin. The "in-vivo" anti-Xa:IIa ratios were: bemiparin 37.9 (95% CI: 28.0 - 55.3, n = 2) and enoxaparin 16.3 (95% Cl: 12.2 - 23.4, n = 8). Enoxaparin induced a higher release of total TFPI, but not on free TFPI, and a longer prolongation of APTT and TT (E-max) than bemiparin, with no differences between groups on Tp-TmT. Adverse events (one in each group) were mild and transient. Conclusion: Bemiparin 3500 IU showed more anti-Xa activity and higher anti-Xa: anti-IIa relationship than enoxaparin 4000 IU in healthy volunteers. Both treatments were well tolerated.

El desarrollo de inhibidores frente al factor VIII en la hemofilia constituye una grave complicación. Se observa en el 20-30 % de los pacientes con hemofilia moderada y grave. El tratamiento más empleado es la inducción de inmunotolerancia con administración diaria de altas dosis de factor VIII. El 25-35% de los pacientes no responden, presentando hemartrosis frecuentes y riesgo de hemorragia vital. Otra alternativa terapéutica es la inmunosupresión. Recientemente se han empleado con éxito nuevos fármacos en el tratamiento de enfermedades autoinmunes refractarias al tratamiento convencional, como el rituximab que es un anticuerpo monoclonal anti-CD20 dirigido contra los linfocitos B. Presentamos un niño de 7 años de edad, diagnosticado a la edad de 13 meses de hemofilia A grave (nivel de FVIII < 1 UI/dl). Desarrolló un inhibidor tras la primera dosis de factor VIII recombinante con un título de 11,8 U Bethesda (UB). Inició el tratamiento de inmunotolerancia con la administración diaria de 100 UI/kg de factor VIII recombinante a través de un porth-a-cath. El reservorio se colocó con el apoyo de administración de factor VII activado. El nivel del inibidor fluctuó durante los 2 años siguientes entre 0,1 y 29,3 UB, sin clínica hemorrágica significativa. Se cambió el tratamiento sustitutivo a factor VIII humano, con disminución transitoria del inhibidor, que se mantuvo durante 4 años entre 2 y 40 UB. A los 7 años de edad y tras un aumento del inhibidor a 49,8 UB comienzan a aparecer hematomas musculares frecuentes en pantorrillas y antebrazos, que precisan tratamiento con factor VII activado recombinante. Aparte del elevado coste del tratamiento, el paciente y su familia tenían muy limitada su calidad de vida, por la dependencia hospitalaria. Se informó a la familia de la posibilidad del tratamiento con rituximab y tras obtención del consentimiento informado para su uso compasivo, se inició la administración durante 4 semanas de una dosis semanal de rituximab a 375 mg/m2/dosis. Tras la primera dosis, presentó una reacción anafiláctica consistente en prurito, disnea, taquicardia e hipotensión que cedió tras la administración de difenhidramina e hidrocortisona y disminución del ritmo de infusión de rituximab. Los niveles de inhibidor tras las 4 semanas disminuyeron a 1,2 UB y se mantuvieron en valores inferiores a 3 UB durante las 18 semanas siguientes. La cifra total de linfocitos permaneció inalterada en tanto que los linfocitos B fueron indetectables durante este tiempo. Las concentraciones de inmunoglobulinas G, A y M disminuyeron ligeramente, pero permaneciendo en límites normales. No se observó aumento del número de infecciones. La profilaxis con factor VIII se redujo desde 200 UI/kg/ día hasta 50 UI/kg, tres veces por semana. Tras observar un aumento de los linfocitos B, se observó un incremento del inhibidor a 34,4 UB con reaparición de clínica hemorrágica. Se repitió la tanda semanal de rituximab durante 4 semanas, continuando con una administración mensual y espaciando posteriormente el intervalo de administración hasta llegar a cada 12 semanas, con monitorización del nivel de inhibidor. El paciente está libre de clínica hemorrágica y no se ha observado reaparición del inhibidor, siguiendo el tratamiento con administración de una dosis única de rituximab cada 12 semanas. Según nuestra experiencia, el tratamiento con rituximab puede ser eficaz en el tratamiento de inhibidores en la hemofilia grave y tras la dosis inicial, debe ser mantenido con la mínima dosis necesaria para la obtención de la respuesta clínico-biológica deseada.

An asymptomatic, 29-year-old woman was referred to our hospital before surgery because in the basic study of hemostasis she showed a prolonged thrombin time (TT) and a normal reptilase time (RT). She had not received any anticoagulants so, to account for these abnormal results the presence of an inhibitor or a dysfibrinogenemia was suspected. A 1:1 mixture of the patient's plasma with control plasma did not correct the TT. Dysfibrinogenemia was excluded because the defibrinated plasma retained the inhibitory activity when mixed with normal plasma. When 0.02 mg/ml of Protamine Sulphate (a concentration that neutralizes 1 U/mL of heparin in normal plasma) was added to the patient's plasma, the inhibitory activity did not disappear. IgG from the patient and from normal serum was isolated. The patient's IgG was able to prolong the TT of a normal plasma and of a purified fibrinogen. The patient IgG did not impair the catalytic activity of thrombin, because no difference was observed in the hydrolysis of S-2238 by 1 U NIH human thrombin with normal or patient IgG. The time course of the thrombin-mediated fibrinopeptide-release from normal fibrinogen with the patient's IgG, showed a delay in the fibrinopeptide B (FPB) release without affecting the fibrinopeptide A (FPA) release. This patient has an IgG antibody that delays fibrinopeptide B release of fibrinogen.