Lily Ngo, Zining (Nick) Li, Yanghao (Andrew) Chen, Haiyue (Violet)Tian PHM142 Fall 2018 Instructor: Dr. J. Henderson Vitamin K Lily Ngo, Zining (Nick) Li, Yanghao (Andrew) Chen, Haiyue (Violet)Tian October 30th, 2018

Vitamin K Quinone derivative Two main forms: Differ at side chain C3 Vitamin K1 (phylloquinone) in plants Vitamin K2 (menaquinone) from intestinal bacteria Differ at side chain C3 Important role in blood coagulation and calcium homeostasis Oldenburg et al. (2008)

The Vitamin K Cycle Important for recycling Vitamin K γ-glutamyl carboxylase (GGCX) catalyzes: Vitamin K hydroquinone → Vitamin K epoxide Vitamin K hydroquinone acts as an electron donor and is oxidized to Vitamin K epoxide Tie, J., & Stafford D.W. (2016)

The Vitamin K Cycle γ-glutamyl carboxylase (GGCX) also catalyzes: glutamic acid → γ- carboxyglutamic acid Binding through pro-peptide region makes prothrombin a stronger chelator for calcium Oldenburg et al. (2008) Tie, J., Jin, D., Straight, D. L., & Stafford, D. W. (2011)

The Vitamin K Cycle The Vitamin K Cycle Vitamin K-epoxide reductase (VKOR) reduces Vitamin K epoxide back to Vitamin K hydroquinone Through VKOR redox center composed of Cys 132 and Cys 135 Oldenburg et al. (2008) Tie, J., Jin, D., Straight, D. L., & Stafford, D. W. (2011)

Vitamin K-Epoxide Reductase (VKOR) VKOR Redox Center with conserved Cys 132 and Cys 135 OLDENBURG, J. , WATZKA, M. , ROST, S. and MÜLLER, C. R. (2007)

The Vitamin K Cycle NADPH-Quinone Oxidoreductase (NQO1) can also reduce Vitamin K to Vitamin K hydroquinone VKOR and NQO1 inhibited by coumarin type drugs Tie, J., & Stafford D.W. (2016)

Vitamin K’s Role in Blood Clotting Activation of clotting factors Factor 2 (Prothrombin) Factor 7 (Proconvertine) Factor 9 (Christmas Factor) Factor 10 (Stuart-Prower Factor) Activation of anticoagulant proteins Protein C Protein S FUN FACT Hemophilia B, factor 9 deficiency, was first documented in 1952 in a patient with the name Stephen Christmas, hence its name. Shantsila, 2018

Activation of Vitamin K dependent Clotting Factors Zymogen: Need to be activated for them to participate in the coagulation pathway → GGCX: convert glutamic acid residues in the amino-terminal to γ-carboxyglutamic acid residues → Increase affinity for calcium ions to bind and form bridges to phospholipid surfaces, which are essential for the formation of activation complexes Zymogen NH2 terminal Ca++ Ca++ Phospholipid Ca++ Ca++ Activated Clotting Factor

Activation of Anticoagulant Proteins (Protein C and Protein S) Have homologous amino acid sequence for the 1st 40 residues (N-terminal) → Each has 9 - 13 γ-carboxyglutamic acid (Gla) residues on N-terminus → Each requires GGCX and Ca++ activity Therefore, have similar modes of activation Amino Acid Sequences of Vitamin K-dependent clotting factors: All have Gla AA on N- terminus Factor 2 Factor 7 Factor 9 Factor 10 Protein C Protein S Nextprot Database

Drugs Related to Vitamin K Coumarin 4-Hydroxycoumarin Dicoumarol Warfarin

Coumarin First isolated in 1820 from tonka beans and sweet clover. Used in perfume and fabric conditioner. Synthesized in 1868 through Perkin Reaction. Precursor of many Vitamin K antagonists and anticoagulants. Itself has no effect on Vitamin K coagulation. Nagaraju, Kuchana 2017

Coumarin and Its Derivatives Dicoumarol (Anticoagulant) 4-Hydroxycoumarin Phenprocoumon (Anticoagulant) Warfarin (Anticoagulant) Acenocoumarol (Anticoagulant)

4-Hydroxycoumarin and Dicoumarol 4-Hydroxycoumarin as a fungal metabolite from coumarin, and further fermentation produces Dicoumarol. Dicoumarol is a natural anticoagulant. Dicoumarol was used medicinally until mid 1950s when warfarin was synthesized. Dicoumarol anticoagulant pathway involves two enzymes: VKOR (Vitamin K Epoxide Reductase) NQO1 (NADPH Quinone Oxidoreductase) Timson, 2017

Dicoumarol and NQO1: Mechanism of action NQO1’s major role in the metabolism (detoxification): catalyzing the NADPH- dependent reduction of various xenobiotics, preventing the build up of reactive oxygen species. Data shown catalysis of menadione (vitamin K derivative) reduction -- unclear mechanism. But NQO1 is not a major enzyme (like VKOR) for Vitamin K reduction → NQO1 knocked down mice can still produce reduced Vitamin K. Dicoumarol will stack (bind) onto the FAD cofactor groups in both dimers. Dicoumarol as a competitive inhibitor to NQO1, which displays negative cooperativity (mutation). Timson, 2017

Dicoumarol and VKOR: Mechanism of Action In γ-carboxylation (activation of clotting factor), Vitamin K acts as a cofactor and is oxidized. VKOR converts oxidized Vitamin K back to normal form. Dicoumarol as an antagonist of VKOR and inhibits oxidized Vitamin K → normal form. Low level of normal Vitamin K → prevents the activation (γ-carboxylation) of clotting factor. Same mechanism for Warfarin Dicoumarol binding on VKOR Binding site Timson, 2017

Warfarin and VKOR Enzyme γ-carboxyglutamyl carboxylase (GGCX) facilitates the conversion of glutamate to γ- carboxyglutamate on clotting factors, with cofactor reduced Vitamin K. Activated clotting factor released to bind platelets. γ-carboxylation causes reduced vitamin K to be oxidized (VK>O), which is then reduced to Vitamin K quinone (normal) by VKOR. VK reduced by several enzymes (including VKOR) to VKH2 that can involve in carboxylation again. With warfarin, amount of reduced VK decreases (most in oxidized state), un-carboxylated clotting factors (7,9,10) released, unable to produce sufficient thrombins → insufficient cross-linked fibrin. Sufficient dietary Vitamin K can bypass the VKOR-inhibited pathway. Incomplete blockage: several other enzymes other than VKOR can reduce VK. Shearer, Okano 2018

Vitamin K in Calcium Homeostasis Vascular Health Bone Metabolism Gröber, U., Reichrath, J., Holick, M. F., & Kisters, K. (2014)

Vitamin K and Vascular Health Matrix Gla protein (MGP) inhibits vascular calcification. MGP is activated by Vitamin K through carboxylation. MGP is upregulated with the development of calcification. It also causes local deficiency of Vitamin K (negative feedback). Non-carboxylated (inactive) MGP is produced. Dp-ucMGP is released into circulation and is correlated with cardiovascular morbidity. El Asmar, M. S., Naoum, J. J., & Arbid, E. J.(2014) Liabeuf, S., Olivier, B., Vemeer, C., Theuwissen, E., Magdeleyns, E., Aubert, C. E.& Massy, Z. A. (2014)

Vitamin K and Vascular Health Vitamin K2 shows an important role in reducing vascular calcification. Vitamin K2 can increase HDL levels and decrease cholesterol levels. El Asmar, M. S., Naoum, J. J., & Arbid, E. J.(2014)

Vitamin K and Bone Metabolism The expression of cytokines(such as OPG) and the expression of RANKL is inhibited. Increase the alkaline phosphatase activity can increase the formation of the organic matrix and mineralization of the bone. Vitamins K2 stimulates SXR to regulate the transcription of osteoblastic biomarker genes and ECM-related genes. Prevents osteoclast formation directly and indirectly. Solmaz A, Amir ,A, Rasouli-G (2018)

Summary Vitamin K is a quinone derivative with two main forms: Vitamin K1 and Vitamin K2 The Vitamin K cycle is important for recycling Vitamin K and converting glutamic acid to γ-carboxyglutamic acid γ-glutamyl carboxylase (GGCX) and Vitamin K-epoxide reductase (VKOR) are two important enzymes in the Vitamin K cycle Vitamin K plays an important role in blood clotting Examples of Vitamin K dependent clotting factors & proteins: Factor 2, 7, 9, 10 & Protein C, S GGCX activates these zymogens by converting GLU residues on N terminus to GLA Vitamin K2 shows an important effect in reducing vascular calcification Vitamin K can facilitate osteoblast function and prevent the apoptosis of osteoblast Only reduced Vitamin K can help with the activation of clotting factors Warfarin inhibits the formation of cross linked fibrin by inhibiting the conversion of oxidized Vitamin K to the reduced form of Vitamin K Dietary intake of Vitamin K can compensate the loss of reduced Vitamin K due to warfarin

References Akbari, S., & Rasouli-Ghahroudi, A. A. (2018). Vitamin K and Bone Metabolism: A Review of the Latest Evidence in Preclinical Studies. BioMed research international, 2018. Combs, G., & McClung, J (2017). Chapter 9 - Vitamin K. The Vitamin (Fifth Edition). Fundamental Aspects in Nutrition and Health. (pp. 243-265. Elsevier Inc. De Vilder, E. et al. (2017) GGCX-Associated Phenotypes: An Overview in Search of Genotype-Phenotype Correlations. INT J MOL SCI, 18 (240). Elsevier Inc. El Asmar, M. S., Naoum, J. J., & Arbid, E. J. (2014). Vitamin K dependent proteins and the role of vitamin K2 in the modulation of vascular calcification: a review. Oman medical journal, 29(3), 172. Gröber, U., Reichrath, J., Holick, M. F., & Kisters, K. (2014). Vitamin K: an old vitamin in a new perspective. Dermato-endocrinology, 6(1), e968490. Nagaraju, G., & Kuchana, V. (2017). A REVIEW ON BIOLOGICAL ACTIVITY AND SYNTHESIS OF COUMARINS. INDO AMERICAN JOURNAL OF PHARMACEUTICAL SCIENCES,4(10), 3510-3527. Nextprot. (2018, September 3). Retrieved October 27, 2018, from https://www.nextprot.org/ Oldenburg, J., Marinova, M., Muller-Reible. C., & Watzka, M. (2008). The Vitamin K Cycle in Litwack, G.’s Vitamin K Volume 78 (pp. 35-62). Elsevier Inc. OLDENBURG, J. , WATZKA, M. , ROST, S. and MÜLLER, C. R. (2007), VKORC1: molecular target of coumarins. Journal of Thrombosis and Haemostasis, 5: 1-6. Rizzetto, M., & Postores, S. (2007). Textbook of hepatology: From basic science to clinical practice. Retrieved October 27, 2018, from http://www.blackwellpublishing.com/content/BPL_Images/Content_store/Sample_Chapter/9781405127417/9781405127417.pd Shantsila, E. (2016). Figure 1.1, [The coagulation cascade]. - Non-Vitamin K Antagonist Oral Anticoagulants - NCBI Bookshelf. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK500197/figure/ch1.Fig1/

References (Page 2) Shearer, M. J., & Okano, T. (2018). Key Pathways and Regulators of Vitamin K Function and Intermediary Metabolism. Annual Review of Nutrition,38(1), 127-151. Stafford, D. W. (2005). The vitamin K cycle. Journal of Thrombosis and Haemostasis, 3: 1873-1878. Tie, J., & Stafford D.W. (2016). Structural and functional insights into enzymes of the vitamin K cycle. J Thromb Haemost, 14: 236–47. Tie, J., Jin, D., Straight, D. L., & Stafford, D. W. (2011). Functional study of the vitamin K cycle in mammalian cells. Blood, 117(10), 2967-2974. Timson, D. (2017). Dicoumarol: A Drug which Hits at Least Two Very Different Targets in Vitamin K Metabolism. Current Drug Targets,18(5), 500-510.