results therefore suggest that an additional reductase supports carboxylation during embryogenesis. A quinone reductase that reduces K to KH 2 is present in adult mouse liver (7). The carboxylase converts KH 2 to KO that the quinone reductase cannot recycle, and vitaminK levels from maternal transfer are low (17). Consequently, a quinone reductase could only support a limited amount of VKD protein carboxylation. In this regard, it is worth noting that many VKD clotting factors also function in signal- ing, activating protease-activated receptors (PARs) thought to be important to embryonic development (18). PT, factor X, and PAR1 deficiency in mice all result in approximately half of the embryos dying midgestation (18–20), which may reflect the role of these VKD proteases in signaling. The amounts of carboxylated protein required for signaling would be substantially less then that needed for clotting, and so a quinone reductase could be functionally significant. We note that quinone reductase activity in adult liver only supports blood clotting in the context of administration of high doses of vitaminK (7, 21), which were not used here; therefore, the presence of the quinone reductase in embryonic liver should not affect blood coagulation and the conclusion of the current study.
Neonatal RDSs are characterized by leakage of plasma proteins of varying sizes into the airspace, which leads to interstitial and intra-alveolar thrombin generation with subsequent fibrin deposition (5), systemic activation of clotting, complement and polymorph nuclear lymphocytes (14) . PTZ, which is a vitaminkdependentprotein, proved to play a role in the prevention of coagulation (6)
Bone that was virtually depleted of the vitaminK-dependentprotein, osteocalcin, and 93% reduced in the concentration of its characteristic amino acid, gamma-carboxyglutamic acid, was obtained from rats treated with warfarin for 6 wk. Osteocalcin-deficient bone particles were resistant to resorption when implanted subcutaneously in normal rats. The relative resorption was 60% of control bone, as measured by histomorphometry as percent of bone particles in the field. Additionally, the number of multinucleated cells around the bone particles was reduced by 54%. These data suggest that osteocalcin is an essential
Introduction: Growth arrest-specific gene 6 protein (GAS6) and protein S (ProS) are vitaminK-dependent proteins present in plasma with important regulatory functions in systems of response and repair to damage. They interact with receptor tyrosine kinases of the Tyro3, Axl and MerTK receptor tyrosine kinase (TAM) family, involved in apoptotic cell clearance (efferocytosis) and regulation of the innate immunity. TAM-deficient mice show spontaneous lupus-like symptoms. Here we tested the genetic profile and plasma levels of components of the system in patients with systemic lupus erythematosus (SLE), and compare them with a control healthy population. Methods: Fifty SLE patients and 50 healthy controls with matched age, gender and from the same geographic area were compared. Genetic analysis was performed in GAS6 and the TAM receptor genes on SNPs previously identified. The concentrations of GAS6, total and free ProS, and the soluble forms of the three TAM receptors (sAxl, sMerTK and sTyro3) were measured in plasma from these samples.
Our results indicate, for the first time, that miR-133a exerts a novel regulatory mechanism on the expression of the main genetic factor on coumarins dose require- ments, VKORC1. After the in silico identi- fication of potential miRNAs involved in VKORC1 expression (miR-133a and miR- 137), also suggested in a previous report (15), we showed that only miR-133a was coexpressed in human healthy livers with VKORC1, which enables this molecule to exert a potential biological effect on VKORC1 levels in the liver. These results are in accordance with the data from the miRNA array published by Tzur et al. (16). We next checked and quantified this possibility in HepG2 cells that constitu- tively express VKORC1. The results showed that miR-133a significantly de- creased VKORC1 mRNA levels in a dose- dependent manner. Additionally, we were also able to observe a decrease of endoge- nous VKORC1 in HepG2 cells transfected with miR-133a. In an attempt to study the nature of the interaction between miR- 133a and VKORC1 in greater depth, the entire 3′UTR of VKORC1 was subcloned in a reporter system downstream of lu- ciferase, and results showed an effect for precursors of miR-133a (see Figure 4A). However, it was published that the 3′UTR from a target mRNA attached to luciferase mRNA could create secondary structures that may be different from the physiologi- cal target mRNA framework (17). To fur- ther confirm our previous results, a vector Figure 4. Reporter studies of the direct VKORC1 mRNA-miRNA interaction. (A) pMIR-
Low vitaminK status (indicated by undercarboxylated MGP) is associated with increased vascular calci ﬁ cations, and these levels can be improved by effective vitaminK supplementation 28–32 It was long believed that vitaminK was only involved in forming coagulation factors (ie, maintaining haemostasis). However, other vitamin-Kdependent proteins (containing γ -carboxyglutamate or Gla) are dependent on vitamin-K carboxylation for func- tionality. 33 VitaminK acts as a cofactor in the conversion of glutamate into Gla. Gla-containing proteins (MGP and osteocalcin) regulate many anticalci ﬁ cation and bone-forming processes in the body, which are depend- ent on vitaminK in order to be produced. Low levels of vitaminK impair activation of osteocalcin and decrease the activity of osteoblasts (cells important for building bone). 33 34 Thus, vitaminK is vital to the functionality of proteins such as osteocalcin (important for building bone), (MGP, the most potent arterial calci ﬁ cation inhibitor known) and the growth-arrest sequence-6 protein (Gas6, involved in cell growth regulation 35
Citrate blood samples for coagulation profile analyses were collected together with the routine laboratory testing for blood count, C-reactive protein (detection limit 0.2 mg/l) and interleukin 6 within the first hour of life prior to vita- min K administration (0.2 mg phytomenadione solution intravenously). Determined components of the coagulation profile included activities of clottable fibrinogen, activity of coagulation factors II, V, VII, VIII, and X, as well as anti- thrombin. Coagulation parameters were analyzed in the platelet poor plasma after centrifugation, the remaining sediment was used for DNA extraction. Details on blood sampling and laboratory methods used to determine con- centrations and activities of the above-mentioned coagula- tion parameters are described elsewhere .
Hereditary combined vitaminK-dependent clotting factors deficiency (VKCFD) is a rare congenital bleeding disorder resulting from variably decreased levels of coagulation factors II, VII, IX and X as well as natural anticoagulants protein C, protein S and protein Z. The spectrum of bleeding symptoms ranges from mild to severe with onset in the neonatal period in severe cases. The bleeding symptoms are often life-threatening, occur both spontaneously and in a surgical setting, and usually involve the skin and mucosae. A range of non-haemostatic symptoms are often present, including developmental and skeletal anomalies. VKCFD is an autosomal recessive disorder caused by mutations in the genes of either gamma-glutamyl carboxylase or vitamin K2,3-epoxide reductase complex. These two proteins are necessary for gamma-carboxylation, a post-synthetic modification that allows coagulation proteins to display their proper function. The developmental and skeletal anomalies seen in VKCFD are the result of defective gamma-carboxylation of a number of non-haemostatic proteins. Diagnostic differentiation from other conditions, both congenital and acquired, is mandatory and genotype analysis is needed to confirm the defect. VitaminK administration is the mainstay of therapy in VKCFD, with plasma supplementation during surgery or severe bleeding episodes. In addition, prothrombin complex concentrates and combination therapy with recombinant activated FVII and vitaminK supplementation may constitute alternative treatment options. The overall prognosis is good and with the availability of several effective therapeutic options, VKCFD has only a small impact on the quality of life of affected patients.
blood coagulation factors 2, 3 . Despite extensive efforts, the components of the VKOR complex have not been identified 4, 5, 6, 7, 8 . The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2), and resistance to coumarin- type anticoagulant drugs (warfarin resistance, WR). Here, we identify, by using linkage information from three species, the gene vitaminK epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Over- expression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition 16 .
The marked homology of the mammalian carboxylases sug- gests the importance of vitaminK and the synthetic machinery to generate ␥ -carboxyglutamic acid. The evolutionary conser- vation of this complex enzymatic system requiring exogenous vitaminK or a vitaminK-like cofactor and multiple enzymes for vitaminK transport and metabolism suggests a critical role for ␥ -carboxyglutamic acid that has been retained throughout phylogeny. ␥ -Carboxyglutamic acid is important for calcium binding and plays a critical functional role in generating the membrane binding properties of the vitaminK-dependent blood clotting proteins (24). The role of ␥ -carboxyglutamic acid in other mammalian proteins such as osteocalcin and matrix Gla protein remains uncertain. The role of ␥ -carboxyglutamic acid in the conotoxins also remains speculative, despite the determination of the structures of some of these peptides (21, 22, 29). We suspect that ␥ -carboxyglutamic acid plays a broader role than the highly specialized activities associated with mem- brane interaction. Perhaps, ␥ -carboxyglutamic acid is critical for function of certain housekeeping proteins that are ubiqui- tous in animal cells, although it would appear that vitaminK-dependent carboxylation is not a required step until late in embryogenesis. 1 Isolation of novel ␥ -carboxyglutamic acid-con-
atherosclerotic plaque. Calcified valves including specimens with calcific aortic stenosis, calcified porcine xenograft valves, and a calcified aortic homograft valve were analyzed for Gla content, complete amino acid analysis, and tissue calcium and phosphorus levels. Normal porcine valves contained protein-bound Gla (2.0-10.6 Gla/10 4 amino acids): no Gla was present in normal valve leaflets. Furthermore, Gla levels paralleled tissue calcium content in the calcified valves. In addition, complete amino acid analysis indicated a decline in valvar collagen content plus increased acidic proteins in conjunction with valvar
Other side effects of warfarin use include; warfarin necrosis in patients deficient in protein C (Chan et al., 2000), purple toe syndrome (Talmadge and Spyropoulos, 2003), osteoporosis has also been seen to likely be a result of the warfarin side effect. as seen in three studies in 1999 (Caraballo et al., 1999), 2002 (Pilon et al., 2004) and 2006 (Gage et al., 2006). Several studies have implicated warfarin use in vascular and valvularcalcification (Palaniswamy et al., 2011). A rarely talked about side effect is calcification of cartilages, blood vessels and even heart valves. Any suggestion that warfarin might be associated with vascularcalcification raises the question as to why so many people, when placed on warfarin, do not develop vascularcalcification. Perhaps the complexity of the vitaminK-dependent carboxylation process might explain why some patients may be at higher risk for the development of associated vascularcalcification. Ultimately, these observations raise questions about whether the risk of vascularcalcification should be added to the risk of bleeding when considering whether to initiate certainpatients on warfarin. Interestingly, patients anticoagulated for peripheralvascular disease had an almost 10 times higher risk of bleeding than those anticoagulated for other reasons (Anand et al., 2007). It is intriguing to wonder whether certainpatients have global underactivity of their carboxylation processes, such as those with undetected vitaminK deficiency, and if suchpatients develop premature vascular disease and/or higher rates of bleeding complications when placed on warfarin. Although this is purely speculative at this point, it raises interesting questions about whether there might be certainidentifiablepopulations that are particularly at risk
Pharmacovigilance practices were implemented at our center (Hospital de Clínicas de Porto Alegre, state of Rio Grande do Sul, Brazil) in 2002 and have since been carried out by the Clinical Pharmacy Service. The hospital’s Drug Information Center is in charge of all such activities, and conducts drug utilization studies and active case-finding of adverse drug reactions (ADRs) with the primary objective of fostering rational use of medicines. The present descriptive study, conducted from June through August 2012, included patients who were prescribed intravenous, intramuscular, or subcutaneous vitaminK. Patients as were neonates (due to routine prophylactic administration of vitaminK) . Patients were identified by means of daily reports of patient prescriptions, issued by the hospital electronic medical records (EMR) system, and by means of active case-finding, which consisted of a daily review of the EMR and contact with staff physicians. The identified patients were followed until discontinuation of vitaminK therapy or until hospital discharge so as to assess the profile of vitaminK prescription at our center. VitaminK was selected as a trigger for identification of adverse events (AEs) associated with excessive use or insufficient monitoring of therapy with oral anticoagulants, such as warfarin [4,5]. All AEs thus identified were treated as ADRs, as all therapeutic doses were individualized. Patients on oral anticoagulant therapy were monitored by means of the prothrombin time (PT), as expressed by the International Normalized Ratio (INR) (reference range 2–3); INR values >4 were flagged due to risk of bleeding and/or hemorrhage. The Naranjo et al. algorithm was used to determine the probability of a causal relationship between the prescribed medications and the suspected ADR, which was classified as definite, probable, possible, or doubtful . ADRs were further classified by predictability as predictable or unpredictable, as per Thompson & Rawlins ; and by severity
In hoofdstuk 5 bestudeerden wij of een dagelijkse toediening van een geringe hoeveelheid vitamine K de stabiliteit van antistollingsbehandeling kan verbeteren. In een experimentele studie werden patiënten willekeurig ingedeeld in een groep die naast de antistollingsbehandeling een supplement met 100 µg vitamine K innamen of in een placebogroep. De keuze voor de dosering vitamine K werd genomen naar aanleiding van een vooronderzoek dat wordt beschreven in hoofdstuk 4. In dit hoofdstuk bepaalden we wat het effect is van verschillende doseringen vitamine K op de benodigde dosis van het antistollingsmiddel om zodoende de dosering vast te stellen die hoog genoeg is om het gewenste effect te bereiken, maar die wel veilig kan worden gegeven. In de experimentele studie vonden wij dat de kwaliteit van antistollingsbehandeling, uitgedrukt als de tijd dat de INR binnen het streefgebied is (time in therapeutic range [TTR]), verbeterde van 85.5% in de placebo groep naar 89.5% in de groep die vitamine K kreeg.
normalize coagulation within one hour (62.2 versus 9.6%; P <0.05) . The role of vitaminK in association with PCC, in maintaining the normalized coagulation over six hours has been emphasized . The importance of rapid hemorrhage control has led international guidelines to recommend the use of PCC rather than FFP [10-12]. For complete VKA reversal, all guidelines recommend an infusion of PCC (at least 20 IU/kg factor IX equivalent) related to admission INR value in combination with at least 5 mg of vitaminK to rapidly achieve a post-reversal INR ≤1.5 and maintain a normal coagulation profile over six hours. So as to save time, French guidelines suggest, on the basis of a previous study, the possibility of adminis- tering a probabilistic single-regimen dose of 25 IU/kg of PCC and 10 mg of vitaminK as soon as a severe hemorrhage is diagnosed [9,10]. In all situations, post- reversal INR must be measured 30 minutes after the infusion to evaluate the efficacy of the treatment and make any necessary adjustments thereafter, and measured six hours later to control the efficacy of vitaminK [10-12]. French guidelines were published in 2008 and have become a standard of care for the management of these patients in France . Treatment time frames were not specified in the international guidelines and the effect of early reversal on mortality has not been studied. At a time when new oral anticoagulant agents without specific antidotes are emerging as promising alternatives to VKAs, it appears important to better define the prognosis benefit of anti- coagulant reversal in the management of patients on oral anticoagulants with severe hemorrhage .
noic acid, but allows it to be reduced into retinol by the microsomal retinol dehydrogenase (Napoli 1996, 1999). Retinol formed by hydrolysis of retinyl esters in the intestinal lumen during uptake also complexes with retinol-binding protein. The protein-retinol complex then serves as a substrate for the conversion of retinol into retinyl esters. The retinyl esters (predominantly but not exclusively retinyl palmitate) are incorporated into chylomicrons, along with triacylglycerols, cholesteryl esters, carotenoids, and other fat-soluble vitamins, secreted into lymph, and transported to the livers for storage. Once in the hepatocytes (retinyl esters are also stored in the lungs and bone marrow), reti- nyl esters undergo hydrolysis to release free retinol, which then binds with retinol-binding protein. This complex is mostly transferred to the hepatic stellate cells and retinol is reesterified. Mobilisation of retinol from these cells involves ester hydrolysis and complexation of free retinol with retinol-binding protein before excretion into plas- ma. Inside the target cells, retinol (having no direct known biological activity) is converted into hormonally active products, e.g. retinal and retinoic acid.
crystallization experiments were purchased from GenScript. FXI was con- centrated to 8 mg/mL and added to an equal volume of peptide sequence YPRHIYPDFPTDTT (P39), FNPISDFPDTTS (HKP), or RLEFPDFPIDD (LP2) to achieve a 1:5 protein-to-peptide molar ratio for P39 and a 1:6 ratio for peptides HKP and LP2. The resulting solution was 4 mg/mL FXI in 50 mM Tris-HCl pH 7.6 and 75 mM NaCl. Crystals were grown in sitting drop from conditions of 0.1 M 2-(N-morpholino)ethanesulfonic acid pH 6.5 and 20% polyethylene glycol 4000 for the FXI-P39 complex and 0.1 M N-2- hydroxyethylpiperazine-N9-2-ethanesulfonic acid pH 7.5 and 20% poly- ethylene glycol 1500 for the FXI-HKP and FXI-LP2 complexes. Data were collected from 2 crystals for FXI-P39 and on a single crystal for FXI-HKP and FXI-LP2 at the Diamond Light Source synchrotron using beamline I04. Data were processed with XDS 20 and reduced in space group P4 3 2 1 2 using the CCP4