Low antithrombin levels are associated with low risk of cardiovascular death but are a risk factor for cancer mortality.

BACKGROUND: Thrombosis is common in subjects suffering from cardiovascular diseases (CVD) and cancer. Hypercoagulation plays a pivotal role in the pathophysiology of thrombosis. Therefore, the inactivation of thrombin, the key enzyme in coagulation, is tightly regulated via antithrombin (AT). AT deficiency is related to thrombosis and cardiovascular death. In this study we investigated the association between AT levels and mortality, in particularly cardiovascular-related and cancer-related death in the general population. METHODS: We studied the association of AT levels and mortality in a prospective cohort sampled from the general Italian population (n = 19,676). AT levels were measured in the baseline samples, and mortality was recorded during a median follow-up period of 8.2 years. Cox regression was performed to investigate the association of all-cause, CVD-related and cancer-related mortality with variations in AT levels. RESULTS: In total, 989 subjects died during follow-up, of which 373 subjects of CVD and 353 of cancer-related causes. Cox analysis revealed that, after adjustment for age, sex, current smoking, BMI, diabetes, hypertension, hypercholesterolemia, history of cardiovascular disease, history of cancer, vitamin K antagonists, antiplatelet medication, heparin and oral contraceptives AT levels were not associated with all-cause mortality (HRQ1vsQ5: 0.92, 95% CI:0.74-1.15). Interestingly, the risk of CVD-related mortality was reduced in subjects with low AT levels compared to subjects with higher AT levels, after adjustment for age and sex and other confounders did not change the association (HRQ1vsQ5: 0.64, 95% CI:0.44-0.91). Moreover, low AT levels were associated with increased cancer mortality in a fully adjusted model (HRQ1vsQ2-5: 1.26, 95% CI:0.88-1.81). CONCLUSIONS: Low AT levels are associated to a lower risk of fatal cardiovascular events in the general population, regardless of age, sex and medication use. In contrast, low AT levels are associated with lower cancer survival. For the first time we show that AT levels lower than the normal range in the general population, even before the development or diagnosis of cancer, are associated with an elevated risk of cancer death.

Octa-arginine boosts the penetration of elastin-like polypeptide nanoparticles in 3D cancer models.

Elastin-like polypeptide (ELP) nanoparticles are a versatile platform for targeted drug delivery. As for any type of nanocarrier system, an important challenge remains the ability of deep (tumor) tissue penetration. In this study, ELP particles with controlled surface density of the cell-penetrating peptide (CPP) octa-arginine (R8) were created by temperature-induced co-assembly. ELPs formed micellar nanoparticles with a diameter of around 60 nm. Cellular uptake in human skin fibroblasts was directly dependent on the surface density of R8 as confirmed by flow cytometry and confocal laser scanning microscopy. Remarkably, next to promoting cellular uptake, the presence of the CPP also enhanced penetration into spheroids generated from human glioblastoma U-87 cells. After 24 h, uptake into cells was observed in multiple layers towards the spheroid core. ELP particles not carrying any CPP did not penetrate. Clearly, a high CPP density exerted a dual benefit on cellular uptake and tissue penetration. At low nanoparticle concentration, there was evidence of a binding site barrier as observed for the penetration of molecules binding with high affinity to cell surface receptors. In conclusion, R8-functionalized ELP nanoparticles form an excellent delivery vehicle that combines tunability of surface characteristics with small and well-defined size.

Salivary tissue factor induces thrombin generation in a diurnal rhythm.

BACKGROUND: Upon tooth extraction, extravascular tissue factor (TF) initiates coagulation to arrest bleeding. Additionally, saliva is in constant contact with the wound and contains extracellular vesicle-derived procoagulant TF. Since the duration of postextraction bleeding is highly variable between patients, we hypothesized this may be caused by variation in saliva-derived TF-induced clotting activity. OBJECTIVES: We aimed to assess the variability of saliva-induced thrombin generation (TG) in healthy individuals. METHODS: TG was performed according to the calibrated automated thrombinography (CAT) method. Diluted saliva was added (instead of recombinant TF and phospholipids [PL]) to normal pooled plasma (NPP) in the absence/presence of anti-TF antibodies. Saliva was collected from healthy individuals in the morning, afternoon and evening. RESULTS: Addition of saliva to NPP induced TG curves similar to those induced by r-TF and PL. Moreover, addition of anti-TF antibodies abolished saliva-induced TG, indicating TF-dependence. A large inter-individual variability (peak CV 31%, range 73-220 nmol/L thrombin) in saliva-induced TG was observed. Interestingly, within subjects, saliva-induced TG was significantly (P = 0.009) increased in the morning (167 ± 40 nmol/L thrombin) compared to the afternoon (124 ± 39 nmol/L thrombin) and evening (123 ± 38 nmol/L thrombin). This diurnal variation was not attributable to gingival stimulation or damage induced by tooth brushing. CONCLUSIONS: We identified a diurnal rhythm in salivary TF activity that may have implications for tooth extraction and dental surgery, as performing invasive procedures in the morning may be beneficial for rapid coagulation. Future studies should correlate salivary TF to clinical outcome (ie, postextraction bleeding) and assess a possible relation with bacterial status in the oral cavity.

Studying disorders of vertebrate iron and heme metabolism using zebrafish.

Iron is a crucial component of heme- and iron-sulfur clusters, involved in vital cellular functions such as oxygen transport, DNA synthesis, and respiration. Both excess and insufficient levels of iron and heme-precursors cause human disease, such as iron-deficiency anemia, hemochromatosis, and porphyrias. Hence, their levels must be tightly regulated, requiring a complex network of transporters and feedback mechanisms. The use of zebrafish to study these pathways and the underlying genetics offers many advantages, among others their optical transparency, ex-vivo development and high genetic and physiological conservations. This chapter first reviews well-established methods, such as large-scale mutagenesis screens that have led to the initial identification of a series of iron and heme transporters and the generation of a variety of mutant lines. Other widely used techniques are based on injection of RNA, including complementary morpholino knockdown and gene overexpression. In addition, we highlight several recently developed approaches, most notably endonuclease-based gene knockouts such as TALENs or the CRISPR/Cas9 system that have been used to study how loss of function can induce human disease phenocopies in zebrafish. Rescue by chemical complementation with iron-based compounds or small molecules can subsequently be used to confirm causality of the genetic defect for the observed phenotype. All together, zebrafish have proven to be - and will continue to serve as an ideal model to advance our understanding of the pathogenesis of human iron and heme-related diseases and to develop novel therapies to treat these conditions.

Second international round robin for the quantification of serum non-transferrin-bound iron and labile plasma iron in patients with iron-overload disorders.

Non-transferrin-bound iron and its labile (redox active) plasma iron component are thought to be potentially toxic forms of iron originally identified in the serum of patients with iron overload. We compared ten worldwide leading assays (6 for non-transferrin-bound iron and 4 for labile plasma iron) as part of an international inter-laboratory study. Serum samples from 60 patients with four different iron-overload disorders in various treatment phases were coded and sent in duplicate for analysis to five different laboratories worldwide. Some laboratories provided multiple assays. Overall, highest assay levels were observed for patients with untreated hereditary hemochromatosis and ß-thalassemia intermedia, patients with transfusion-dependent myelodysplastic syndromes and patients with transfusion-dependent and chelated ß-thalassemia major. Absolute levels differed considerably between assays and were lower for labile plasma iron than for non-transferrin-bound iron. Four assays also reported negative values. Assays were reproducible with high between-sample and low within-sample variation. Assays correlated and correlations were highest within the group of non-transferrin-bound iron assays and within that of labile plasma iron assays. Increased transferrin saturation, but not ferritin, was a good indicator of the presence of forms of circulating non-transferrin-bound iron. The possibility of using non-transferrin-bound iron and labile plasma iron measures as clinical indicators of overt iron overload and/or of treatment efficacy would largely depend on the rigorous validation and standardization of assays.