Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria.

Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in CLPX inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism.

Effects of Repeated Bouts of Exercise on the Hemostatic System.

Physical activity is beneficial for health, for example, by lowering the risk of cardiovascular events. However, vigorous exercise is associated with the occurrence of thromboembolic events and sudden cardiac death, in particular in untrained individuals. Whereas acute exercise is known to cause a hypercoagulable state, repeated exposure to (strenuous) exercise by means of training may actually condition the hemostatic response to exercise. To date, the effects of exercise training on blood coagulability and the underlying mechanisms have yet to be fully discerned. In this review, the authors provide an overview of existing literature on how training programs and training status influence hemostasis in healthy individuals. Furthermore, they present data of a pilot study in which we studied the effects of repetitive submaximal intensity cycling on procoagulant and anticoagulant processes. It is known that factor VIII (FVIII) and von Willebrand factor (VWF) increase after exercise, but we found that this increase in FVIII and VWF (antigen, propeptide, and VWF in active conformation) was smaller on each of three subsequent days, suggesting either adaptation of endothelial activation or exhaustion of endothelial VWF supplies. With respect to thrombin generation, elevated FVIII significantly increased the thrombin generation peak but not the endogenous thrombin potential. In contrast, platelet activation in terms of P-selectin expression after stimulation with protease-activated receptor-1 and glycoprotein VI agonists decreased after exercise and did not recover, indicating exhaustion of the platelet response to repetitive exercise.

Toward Worldwide Hepcidin Assay Harmonization: Identification of a Commutable Secondary Reference Material.

BACKGROUND: Absolute plasma hepcidin concentrations measured by various procedures differ substantially, complicating interpretation of results and rendering reference intervals method dependent. We investigated the degree of equivalence achievable by harmonization and the identification of a commutable secondary reference material to accomplish this goal. METHODS: We applied technical procedures to achieve harmonization developed by the Consortium for Harmonization of Clinical Laboratory Results. Eleven plasma hepcidin measurement procedures (5 mass spectrometry based and 6 immunochemical based) quantified native individual plasma samples (n = 32) and native plasma pools (n = 8) to assess analytical performance and current and achievable equivalence. In addition, 8 types of candidate reference materials (3 concentrations each, n = 24) were assessed for their suitability, most notably in terms of commutability, to serve as secondary reference material. RESULTS: Absolute hepcidin values and reproducibility (intrameasurement procedure CVs 2.9%-8.7%) differed substantially between measurement procedures, but all were linear and correlated well. The current equivalence (intermeasurement procedure CV 28.6%) between the methods was mainly attributable to differences in calibration and could thus be improved by harmonization with a common calibrator. Linear regression analysis and standardized residuals showed that a candidate reference material consisting of native lyophilized plasma with cryolyoprotectant was commutable for all measurement procedures. Mathematically simulated harmonization with this calibrator resulted in a maximum achievable equivalence of 7.7%. CONCLUSIONS: The secondary reference material identified in this study has the potential to substantially improve equivalence between hepcidin measurement procedures and contributes to the establishment of a traceability chain that will ultimately allow standardization of hepcidin measurement results.

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.

Performance of the CellaVision DC-1 digital cell imaging analyser for differential counting and morphological classification of blood cells.

AIMS: Recently, a new automated digital cell imaging analyser (Sysmex CellaVision DC-1), intended for use in low-volume and small satellite laboratories, has become available. The purpose of this study was to compare the performance of the DC-1 with the Sysmex DI-60 system and the gold standard, manual microscopy. METHODS: White blood cell (WBC) differential counts in 100 normal and 100 abnormal peripheral blood smears were compared between the DC-1, the DI-60 and manual microscopy to establish accuracy, within-run imprecision, clinical sensitivity and specificity. Moreover, the agreement between precharacterisation and postcharacterisation of red blood cell (RBC) morphological abnormalities was determined for the DC-1. RESULTS: WBC preclassification and postclassification results of the DC-1 showed good correlation compared with DI-60 results and manual microscopy. In addition, the within-run SD of the DC-1 was below 1 for all five major WBC classes, indicating good reproducibility. Clinical sensitivity and specificity were, respectively, 96.7%/95.9% compared with the DI-60% and 96.6%/95.3% compared with manual microscopy. The overall agreement on RBC morphology between the precharacterisation and postcharacterisation results ranged from 49% (poikilocytosis) to 100% (hypochromasia, microcytosis and macrocytosis). CONCLUSIONS: The DC-1 has proven to be an accurate digital cell imaging system for differential counting and morphological classification of WBCs and RBCs in peripheral blood smears. It is a compact and easily operated instrument that can offer low-volume and small satellite laboratories the possibilities of readily available blood cell analysis that can be stored and retrieved for consultation with remote locations.

25-OH Vitamin D concentrations measured by LC-MS/MS are equivalent in serum and EDTA plasma.

In contrast to a recent study reporting an unexpected significant difference for total 25-hydroxyvitamin D (25(OH)D) between serum and EDTA plasma, we demonstrate that concentrations of total 25(OH)D, 25(OH)D2, 25(OH)D3 and 24,25(OH)2D3 do not differ between matched serum and EDTA plasma samples, using two well-characterized LC-MS/MS methods.

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.

Increased BMI and Blood Lipids Are Associated With a Hypercoagulable State in the Moli-sani Cohort.

The coagulation system can be assessed by the thrombin generation (TG) assay, and increased TG peak height, endogenous thrombin potential (ETP), and velocity index are associated with an increased risk of thrombosis. Obesity had been reported to increase TG and is associated with dyslipidemia, which also predisposes to atherosclerotic cardiovascular disease (CVD). However, the effect of the blood lipid profile on TG has not been studied extensively. To gain more insight into the associations of TG, body mass index (BMI) and lipid profile, we studied TG in relation to these parameters in a large Italian population cohort, the Moli-sani study (N = 22,546; age ≥ 35 years; 48% men). TG was measured in plasma samples collected at the enrollment of subjects in the Moli-sani study. TG was triggered with 1 or 5 pM tissue factor, and TG parameters lag time, peak, ETP, time-to-peak (TTP) and velocity index (VI). Additionally, thrombomodulin was added to assess the function of the activated protein C system during TG. In both women and men, overweight (BMI 25-30 kg/m2) and obesity (BMI > 30 kg/m2) were significantly associated with higher ETP, peak and VI (all p < 0.001). High total cholesterol, triglycerides and LDL-cholesterol levels were significantly associated with increased ETP and peak (all p < 0.001). Linear regression analysis revealed that the ETP is positively associated with both plasma LDL and HDL cholesterol levels, whereas the velocity index is positively associated with HDL cholesterol. Additionally, ETP, peak and VI were significantly associated with the plasma triglycerides content. In conclusion, our study shows significant associations of high BMI and blood lipid levels with increased TG parameters, and this hypercoagulability may partly explain the increased risk of CVD in individuals with obesity and/or dyslipidemia.

Implant stability in patients treated with platelet-rich fibrin and bovine bone substitute for alveolar ridge preservation is associated with peripheral blood cells and coagulation factors.

AIMS: The aim of the present study was to assess the association between dental implant stability and peripheral blood cell composition and levels of coagulation factors in patients treated with alveolar ridge preservation with platelet-rich fibrin (PRF) and bovine bone substitute. MATERIALS AND METHODS: Fifty patients were included between 2015 and 2017. PRF was prepared from autologous blood, in which blood cells and coagulation factor levels were measured. PRF and bovine bone were placed in the socket, followed by closure with PRF membrane. Implants were placed 14 (±2.5) weeks postextraction. The implant stability quotient was measured at t = 0, t = 10 days, t = 7 weeks, and t = 17 weeks by resonance frequency analysis. RESULTS: Erythrocyte count was inversely associated with PRF membrane length, but not with implant stability. Conversely, platelet count did not correlate with membrane size but inversely correlated with implant stability at 7 and 17 weeks. In addition, implant stability was directly correlated with levels FXIII (t = 0, p < .01), active von Willebrand factor (VWF; t = 0 and 7 weeks, p < .05), and total VWF (t = 7 weeks, p = .012). CONCLUSION: Implant stability following alveolar ridge preservation with PRF and bovine bone substitute is associated with circulating blood cells and coagulation factors. In particular, fibrin structure, VWF, and FXIII may be important modulators of implant stability.

Acute exacerbations of COPD are associated with a prothrombotic state through platelet-monocyte complexes, endothelial activation and increased thrombin generation.

INTRODUCTION: Patients with chronic obstructive pulmonary disease (COPD) are at increased risk for cardiovascular events, particularly following an acute exacerbation (AE-COPD). Exacerbations are associated with increased systemic inflammation, which may drive coagulation. This prospective cohort study aimed to determine how an AE-COPD affects platelet activation, the endothelium, plasmatic coagulation and fibrinolysis, and its association with systemic inflammation. MATERIALS AND METHODS: Fifty-two patients with an AE-COPD were included. Blood samples at admission, at day 3 of treatment and at convalescence were available for 32 patients. Platelet-monocyte complex (PMC) formation, monocyte Mac-1 expression and platelet (re)activity (P-selectin expression, αIIbß3 activation) were measured by flow cytometry. Von Willebrand Factor (VWF), thrombin generation (TG) and clot lysis time (CLT) were determined as measures of endothelial activation, plasmatic coagulation and fibrinolysis, respectively. RESULTS: Exacerbations were associated with increased PMCs (MFI 31.3 vs 23.8, p = 0.004) and Mac-1 (MFI 38.2 vs 34.8, p = 0.006) compared to convalescence, but not with changes in platelet (re)activity. VWF (antigen, activity, active fraction) and TG (peak, ETP and velocity index) were all significantly higher during AE-COPD compared to convalescence. PMCs, Mac-1, VWF and TG were positively associated with systemic inflammation (CRP). CLT was prolonged in AE-COPD patients with systemic inflammation. Moreover, platelet hyperreactivity on admission was associated with an increased risk for exacerbation relapse. CONCLUSIONS: Acute exacerbations are associated with an inflammation-associated prothrombotic state, characterized by increased PMCs, endothelial activation and plasmatic coagulation. Our findings provide direction for future studies on biomarkers predicting the risk of exacerbation relapse and cardiovascular events.

Platelet Integrin αIIbß3 Activation is Associated with 25-Hydroxyvitamin D Concentrations in Healthy Adults.

BACKGROUND: Cardiovascular events are associated with low circulating vitamin D concentrations, although the underlying mechanisms are poorly understood. This study investigated associations between 25-hydroxyvitamin D concentrations, platelet function, and single-nucleotide polymorphisms (SNPs) in genes influencing vitamin D biology in the 500 Functional Genomics (500FG) cohort. METHODS: In this observational study, platelet activation and function were measured by flow cytometry by binding of fibrinogen to the activated fibrinogen receptor integrin αIIbß3 and expression of P-selectin, markers of platelet aggregation and degranulation, respectively. These parameters were correlated to serum 25-hydroxyvitamin D and genotyping was performed to investigate SNPs in genes important for vitamin D biology. RESULTS: Circulating 25-hydroxyvitamin D concentrations correlated inversely with baseline platelet binding of fibrinogen to integrin αIIbß3 (Pearson's r= -0.172, p = 0.002) and platelet responses to platelet agonist cross-linked collagen-related peptide (CRP-XL) (Pearson's r= -0.196,p = 0.002). This effect was due to circulating vitamin D levels ≤50nmol/L, since no differences in platelet fibrinogen binding were observed between subjects with normal 25-hydroxyvitamin D concentrations (>75nmol/L) and a 25-hydroxyvitamin D insufficiency (50-75 nmol/L). No correlations between 25-hydroxyvitamin D concentrations and platelet P-selectin expression were found. Several SNPs in the GC region of the vitamin D binding proteingene were associated with platelet responses to CRP-XL. CONCLUSION: Low circulating vitamin D concentrations are associated with increased platelet fibrinogen binding to integrin αIIbß3 in unstimulated samples and after stimulation with CRP-XL. These findings may contribute to the increased incidence of cardiovascular events in vitamin D deficient adults and its seasonal variation. Further studies are needed to investigate causality.

A Hypoxic Environment Attenuates Exercise-Induced Procoagulant Changes Due to Decreased Platelet Activation.

Introduction Although physical exercise is protective against cardiovascular disease, it can also provoke sudden cardiac death (exercise paradox). Epidemiological studies suggest that systemic hypoxia at high altitude is a risk factor for venous thromboembolism. Forthcoming, this study investigated the effect of repeated exercise at high altitude on blood coagulation, platelet function, and fibrinolysis. Methods Six trained male volunteers were recruited. Participants ascended from sea level to 3,375 m altitude. They performed four exercise tests at 65 to 80% of their heart-rate reserve during 2 hours: one time at sea level and three times on consecutive days at 3,375 m altitude. Thrombin generation (TG) was measured in whole blood (WB) and platelet-rich and platelet-poor plasma. Coagulation factor levels were measured. Platelet activation was measured as αIIbß3 activation and P-selectin expression. Fibrinolysis was studied using a clot-lysis assay. Results Normoxic exercise increased plasma peak TG through increased factor VIII (FVIII), and increased von Willebrand factor (VWF) and active VWF levels. Platelet granule release potential was slightly decreased. After repetitive hypoxic exercise, the increase in (active) VWF tapered, and there was no more distinct exercise-related increase in peak. Platelet aggregation potential and platelet-dependent TG decreased at high altitude. There were no effects on fibrinolysis upon exercise and/or hypoxia. Conclusion Strenuous exercise induces a procoagulant state that is mediated by the endothelium, by increasing VWF and secondarily raising FVIII levels. After repetitive exercise, the amplitude of the endothelial response to exercise diminishes. A hypoxic environment appears to further attenuate the procoagulant changes by decreasing platelet activation and platelet-dependent TG.

Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor.

BACKGROUND: Interaction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF A1 domain, enabling platelet glycoprotein Ibα binding. Quantification of this ''active" conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction. METHODS: We developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF A1 domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters. RESULTS: Intra- and inter-assay CVs were between 2.4-7.2% and 4.1-9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103±3%. The assay was linear in the range of 90.1-424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6-154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF:GP1bM binding. CONCLUSIONS: We developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.

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.

Effects of Plasmin on von Willebrand Factor and Platelets: A Narrative Review.

Plasmin is the major fibrinolytic protease responsible for dissolving thrombi by cleavage of its primary substrate fibrin. In addition, emerging evidence points to other roles of plasmin: (1) as a back-up for ADAMTS13 in proteolysis of ultra-large von Willebrand factor (VWF) multimers and (2) as an activator of platelets. Although the molecular mechanisms of fibrinolysis are well defined, insights on the effects of plasmin on VWF and platelets are relatively scarce and sometimes conflicting. Hence, this review provides an overview of the literature on the effects of plasmin on VWF multimeric structures, on VWF binding to platelets, and on platelet activation. This information is placed in the context of possible applications of thrombolytic therapy for the condition thrombotic thrombocytopenic purpura.

Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test.

INTRODUCTION: Platelet function testing with flow cytometry has additional value to existing platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine and prediction of thrombosis. The major challenge is to use this technique as a diagnostic test. The aim of this study is to standardize preparation, optimization and validation of the test kit and to determine reference values in a population of 129 healthy individuals. METHODS: Platelet function tests with 3 agonists and antibodies against P-selectin, activated αIIbß3 and glycoprotein Ib (GPIb), were prepared and stored at -20°C until used. Diluted whole blood was added and platelet activation was quantified by the density of activation markers, using flow cytometry. Anti-mouse Ig κ particles were included to validate stability of the test and to standardize results. Reference intervals were determined. RESULTS: Blood stored at room temperature (RT) for up to 4h after blood donation and preheated/tested at 37°C resulted in stable results (%CV<10%), in contrast to measuring at RT. The intra-assay %CV was <5%. Incubation of anti-mouse Ig κ particles with antibodies stored for up to 12 months proved to give a stable fluorescence. The inter-individual variation measured in the 129 individuals varied between 23% and 37% for P-selectin expression and αIIbß3 activation, respectively. CONCLUSIONS: The current study contributes to the translation of flow cytometry based platelet function testing from a scientific tool to a diagnostic test. Platelet function measurements, using prepared and stored platelet activation kits, are reproducible if executed at 37°C. The reference ranges can be validated in clinical laboratories and ongoing studies are investigating if reduced platelet reactivity in patients with bleeding complications can be detected.

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.