In Vivo-Assay for Warfarin-Resistance by Coexpression of VKORC1 and Vitamin K-Dependent Coagulation Factors

2008 ◽  
pp. 259-260
Author(s):  
A. Fregin ◽  
S. Rost ◽  
C. R. Müller ◽  
J. Oldenburg
Keyword(s):  
Vitamin K ◽  
Coagulation Factors ◽  
In Vivo Assay ◽  
Warfarin Resistance

IN VIVO-ASSAY FOR WARFARIN-RESISTANCE BY COEXPRESSION OF VKORC1 AND VITAMIN K-DEPENDENT COAGULATION FACTORS

2007 ◽  
Vol 5 ◽  
pp. P-S-049-P-S-049
Author(s):  
A. Fregin ◽  
M. Taverna ◽  
S. Rost ◽  
C.R. Mueller-Reible ◽  
J. Oldenburg
Keyword(s):  
Vitamin K ◽  
Coagulation Factors ◽  
In Vivo Assay ◽  
Warfarin Resistance

GLA-CONTAINING PROTEINS FROM CALCIFIED HUMAN ATHEROSCLEROTIC PLAQUES

1987 ◽  
Author(s):  
L J M Van Haarlem ◽  
H C Hemker ◽  
B A M Soute ◽  
C Vermeer
Keyword(s):  
Vitamin K ◽  
Vessel Wall ◽  
High Performance ◽  
Gel Filtration ◽  
Coagulation Factors ◽  
Atherosclerotic Plaques ◽  
Factor X ◽  
Carboxylase Activity

Vitamin K-dependent carboxylase activity has been detected in human andbovine vessel wall. Studies comparingthe carboxylases from liver and vessel wall revealed that the enzyme systems may be regarded as isoenzymes withwidely different substrate specificities. The carboxylated product of vessel wall carboxylase has not yet been identified, but it seems plausible that it will be found amongst the Gla-containing proteins which are abundantly present in calcified atherosclerotic plaques (Gla= gammacarboxyglutamicacid, the abnormal amino acid formed by vitamin K-dependent carboxylase). Therefore we have started to characterize the protein constituents of hardened atherosclerotic plaques.The calcified areas from human aortae were solubilized in EDTA and the proteins extracted were partly purified by batch-wise adsorption onto QAE and elution with high salt. The crudeplaque-extract did not contain prothrombin, factor X or protein C. This excludes the possibility that Gla-containing coagulation factors are bound non-specifically from blood. Osteocalcin accounted for 20% of the total amount of protein-bound Gla-residues.Another Gla-containing protein waspurified from the crude plaque-extract by employing high performance liquid chromatography (HPLC). Gel filtration yielded a Gla-rich protein with anapparent Mr of 25 kD. In vitro boththe crude plaque-extract and the purified Gla-containing protein strongly inhibited the precipitation of calcium phosphate and calcium carbonate. A similar effect was not found with humanserum albumin nor with a thermallydecarboxylated plaque-extract. If also in vivo the Gla-containing proteinsproduced by vessel wall carboxylase prevent the precipitation of calcium salts remains to be investigated.

Mutations in the VKORC1 Gene Cause Warfarin Resistance, Warfarin Sensitivity and Combined Deficiency of Vitamin K Dependent Coagulation Factors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 277-277 ◽  
Author(s):  
Johannes Oldenburg ◽  
Simone Rost ◽  
Andreas Fregin ◽  
Christof Geisen ◽  
Vytautas Ivaskevicius ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Vitamin K ◽  
Expression System ◽  
Coagulation Factors ◽  
Wild Type ◽  
Warfarin Resistance ◽  
Cell Expression ◽  
Vkorc1 Gene ◽  
Combined Deficiency ◽  
Cell Expression System

Abstract Coumarins target blood coagulation via inhibition of the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, an essential cofactor for the post-translational gamma-carboxylation of several blood coagulation factors. Recently, two groups including ours identified a key component of the VKOR which we named Vitamin K Epoxid Reductase Subunit 1 (VKORC1). The corresponding gene comprises a 5 kb genomic region and consist of three exons encoding a small 163 aa transmembrane protein. Since VKOR was hypothesized to be involved in two heritable diseases, combined deficiency of vitamin K dependent clotting factors type 2 (VKCFD2) and resistance to coumarin-type drugs (warfarin/phenprocoumon resistance, WR) we sequenced the VKORC1 gene in patients with these phenotypes. The same homozygous missense mutation (R98W) was found in two families from Germany and Lebanon with a mild VKCFD2 phenotype. In 7 patients from 5 families with various degrees of WR (weekly warfarin dose ranged from 115 mg to 280 mg, two patients exhibited complete WR) different heterozygous missense mutations distributed throughout the gene (V29L, V45A, R58G, V66M, L128R) were identified. Furthermore, we proved a heterozygous nonsense mutation (W5Stop) in a young female patient with highly increased phenprocoumon sensitivity. Over-expression of the wild-type protein in HEK 293 cells leads to a striking increase in VKOR activity which is sensitive to warfarin inhibition. In contrast to the wild-type protein, the VKCFD2 variant exhibited a dramatically decrease of VKOR activity. The WR variants showed a varying reduction of the VKOR activity, but unexpectedly, were sensitive to warfarin in the HEK cell expression system The reason for the differing WR phenotypes in human and in the HEK cell expression system are still to be elucidated. In conclusion, VKORC1 protein most likely represents the molecular target of coumarins, which have prescribed for oral anticoagulant therapy since more than 60 years. Mutations in the VKORC1 gene are causative for the hereditary conditions of VKCFD2, warfarin resistance and also warfarin sensitivity. Our findings may provide a basis for a rational design of novel anticoagulants targeting VKOR in the future.

scholarly journals VKORC1L1, An Enzyme Mediating the Effect of Vitamin K in Liver and Extrahepatic Tissues

Nutrients ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 970 ◽  
Author(s):  
Julie Lacombe ◽  
Mathieu Ferron
Keyword(s):  
Vitamin K ◽  
Current Knowledge ◽  
Coagulation Factors ◽  
Original Data ◽  
Quinone Reductase ◽  
Culture Models ◽  
Essential Nutrient ◽  
Partial Redundancy

Vitamin K is an essential nutrient involved in the regulation of blood clotting and tissue mineralization. Vitamin K oxidoreductase (VKORC1) converts vitamin K epoxide into reduced vitamin K, which acts as the co-factor for the γ-carboxylation of several proteins, including coagulation factors produced by the liver. VKORC1 is also the pharmacological target of warfarin, a widely used anticoagulant. Vertebrates possess a VKORC1 paralog, VKORC1-like 1 (VKORC1L1), but until very recently, the importance of VKORC1L1 for protein γ-carboxylation and hemostasis in vivo was not clear. Here, we first review the current knowledge on the structure, function and expression pattern of VKORC1L1, including recent data establishing that, in the absence of VKORC1, VKORC1L1 can support vitamin K-dependent carboxylation in the liver during the pre- and perinatal periods in vivo. We then provide original data showing that the partial redundancy between VKORC1 and VKORC1L1 also exists in bone around birth. Recent studies indicate that, in vitro and in cell culture models, VKORC1L1 is less sensitive to warfarin than VKORC1. Genetic evidence is presented here, which supports the notion that VKORC1L1 is not the warfarin-resistant vitamin K quinone reductase present in the liver. In summary, although the exact physiological function of VKORC1L1 remains elusive, the latest findings clearly established that this enzyme is a vitamin K oxidoreductase, which can support γ-carboxylation in vivo.

scholarly journals Characterizing Coagulation FVII from iPSC-Hepatocytes-like Cells: Setting the Basis for Cell Therapy Development

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4
Author(s):  
Maria Eugenia Chollet ◽  
Elisabeth Andersen ◽  
Maria Eugenia de la Morena-Barrio ◽  
Carlos Bravo-Perez ◽  
Marie-Christine Mowinckel ◽  
...  
Keyword(s):  
Vitamin K ◽  
Thrombin Generation ◽  
Coagulation Factors ◽  
Activity Level ◽  
Primary Hepatocytes ◽  
Post Translational Modifications ◽  
Cellular Expression ◽  
Plasma Pool ◽  
Pngase F

Introduction To date studies on factor (F)VII and other hepatic vitamin K-dependent coagulation factors have relied on cell lines overexpressing these human genes. Even though these models have provided insight into the biology of these factors, they do not fully illustrate the in vivo situation. Thus, a relevant physiological model that mimics the in vivo processing of FVII in liver cells with potential for therapeutic use is needed. Methods Human induced pluripotent stem cells (hiPSCs) were differentiated into hepatocyte-like cells (iHLCs) using a non-transcription factor based, small molecule approach. Cells were grown in medium with vitamin K to ensure a correct gamma-carboxylation. Cellular FVII mRNA and protein were determined by RT-qPCR and proteomic and Western blot (WB), respectively. Secreted FVII antigen was measured by ELISA and WB and FVII activity was assessed by chromogenic assay and thrombin generation assay (TGA). Post-translational modifications of FVII protein (glycosylation) were studied using digestion with N-glycosylase F (PNGase F) and neuraminidase. Confocal immunofluorescence microscopy was used to assess the cellular expression of FVII and other vitamin K- dependent coagulation factors and inhibitors. Human primary hepatocytes or human plasma pool were used as a control in the assays. Results The resulting iHLCs expressed FVII mRNA in comparable levels to primary hepatocytes and cellular FVII peptides were identified by mass spectrometry studies. iHLCs secreted FVII at levels of around 70% compared to primary hepatocytes with detectable activity around 35% of the FVII activity level from primary hepatocytes. The TGA showed that cell medium from iHLCs when mixed with FVII deficient plasma was able to induce thrombin generation faster than the FVII depleted plasma alone (lagtime 3.2 vs 27.6 s, respectively). PNGase-F treatment showed that FVII secreted by iHLCs was N-glycosylated. Intracellular FVII was detected by WB as a band of approximately 63 kDa, slightly larger than FVII from plasma pool but similar to FVII from primary hepatocytes. Moreover, additional coagulation factors and inhibitors such as FII, FX, protein C and antithrombin were detected both at the mRNA and protein levels in the cells. Conclusions Stem cell-derived iHLCs produce and secrete FVII at physiologically relevant levels. The resulting FVII showed similar post-translational modifications to plasma FVII although some differences in proteolysis could be inferred. This iHLCs-derived FVII is able to initiate the extrinsic coagulation pathway. Our data support that these iHLCs can serve as a highly relevant model to study FVII and other vitamin K-dependent coagulation factors in vitro and constitute an important step towards the development of novel cell-based therapies for both FVII and other vitamin K-dependent coagulation factor deficiencies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting of Adenovirus Serotype 5 (Ad5) and 5/47 Pseudotyped Vectors In Vivo: Fundamental Involvement of Coagulation Factors and Redundancy of CAR Binding by Ad5

2007 ◽  
Vol 81 (17) ◽  
pp. 9568-9571 ◽  
Author(s):  
Simon N. Waddington ◽  
Alan L. Parker ◽  
Menzo Havenga ◽  
Stuart A. Nicklin ◽  
Suzanne M. K. Buckley ◽  
...  
Keyword(s):  
Vitamin K ◽  
Kupffer Cell ◽  
Coagulation Factors ◽  
Intravascular Injection ◽  
Adenovirus Serotype ◽  
Liver Targeting ◽  
Serotype 5 ◽  
Binding Capability

ABSTRACT Vitamin K-dependent coagulation factors can promote adenoviral cell transduction in vitro. In vivo, warfarin pretreatment ablates liver targeting of an adenovirus serotype 5 (Ad5) vector deleted of CAR binding capability. Here, we assess in vivo transduction and biodistribution of Ad5 vectors with nonmodified fibers (Ad5) and a serotype 47 fiber-pseudotyped Ad5 (Ad5/47; subgroup D) virus following intravascular injection. Warfarin reduced liver transduction by both viruses. However, no impact on early liver virus accumulation was observed, suggesting no effect on Kupffer cell interactions. Hence, coagulation factors play a pivotal role in selectively mediating liver hepatocyte transduction of Ad5 and Ad5/47 vectors.

Increased Activity of Vitamin K-Dependent Clotting Factors in Human Hyperlipoproteinaemia – Association with Cholesterol and Triglyceride Levels

1977 ◽  
Vol 38 (02) ◽  
pp. 0465-0474 ◽  
Author(s):  
M Constantino ◽  
C Merskey ◽  
D. J Kudzma ◽  
M. B Zucker
Keyword(s):  
Vitamin K ◽  
Plasma Lipids ◽  
Coagulation Factors ◽  
Clotting Factor ◽  
Factor X ◽  
Clotting Factors ◽  
Blood Coagulation Factors ◽  
Cholesterol Levels ◽  
Type V ◽  
Increased Activity

SummaryLevels of blood coagulation factors, cholesterol and triglyceride were measured in human plasma. Prothrombin was significantly elevated in type Ha hyperlipidaemia; prothrombin and factors VII, IX and X in type lib; and prothrombin and factors VII and IX in type V. Multiple regression analysis showed significant correlation between the levels of these plasma lipids and the vitamin K-dependent clotting factors (prothrombin, factors VII, IX and X). Higher cholesterol levels were associated with higher levels of prothrombin and factor X while higher triglyceride levels were associated with higher levels of these as well as factors VII and IX. Prothrombin showed a significant cholesterol-triglyceride interaction in that higher cholesterol levels were associated with higher prothrombin levels at all levels of triglyceride, with the most marked effects in subjects with higher triglyceride levels. Higher prothrombin levels were noted in subjects with high or moderately elevated (but not low) cholesterol levels. Ultracentrifugation of plasma in a density of 1.21 showed activity for prothrombin and factors VII and X only in the lipoprotein-free subnatant fraction. Thus, a true increase in clotting factor protein was probably present. The significance of the correlation between levels of vitamin K-dependent clotting factors and plasma lipids remains to be determined.

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