Calcium ions: Ca2+ binds the negatively charged γ-carboxyglutamate (Gla) residues present in four of the serine proteases of clotting (FII, FVII, FIX, and FX), facilitating the binding of these proteins to exposed phospholipids (Fig. 35.4). The Gla residues are good chelators of Ca2+ because of their two adjacent negatively charged carboxylate groups (Fig. 35.5). [Note: The use of chelating agents such as sodium citrate to bind Ca2+ in blood-collecting tubes or bags prevents the blood from clotting.]
C. Formation of γ-carboxyglutamate residues γ-Carboxylation is a posttranslational modification in which 9–12 glutamate residues (at the amino [N]-terminus of the target protein) get carboxylated at the γ carbon, thereby forming Gla residues. The process occurs in the rough endoplasmic reticulum (RER) of the liver.
1. γ-Carboxylation: This carboxylation reaction requires a protein substrate, oxygen (O2), carbon dioxide (CO2), γ-glutamyl carboxylase, and the hydroquinone form of vitamin K as a coenzyme (Fig. 35.6). In the reaction, the hydroquinone form of vitamin K gets oxidized to its epoxide form as O2 is reduced to water. [Note: Dietary vitamin K, a fat-soluble vitamin (see p. 393), is reduced from the quinone form to the hydroquinone coenzyme form by vitamin K reductase (Fig. 35.7).] 2. Inhibition by warfarin: The formation of Gla residues is sensitive to inhibition by warfarin, a synthetic analog of vitamin K that inhibits the enzyme vitamin K epoxide reductase (VKOR). The reductase, an integral protein of the RER membrane, is required to regenerate the functional hydroquinone form of vitamin K from the epoxide form generated in the γ-carboxylation reaction. Thus, warfarin is an anticoagulant that inhibits clotting by functioning as a vitamin K antagonist. Warfarin salts are used therapeutically to limit clot formation. [Note: Warfarin is used commercially as a pest control agent such as in rat poison. It was developed by the Wisconsin Alumni Research Foundation, hence the name.]
Genetic differences (genotypes) in the gene for catalytic subunit 1 of VKOR (VKORC1) influence patient response to warfarin. For example, a polymorphism (see p. 491) in the promoter region of the gene decreases gene expression, resulting in less VKOR being made, thereby necessitating a lower dose of warfarin to achieve a therapeutic level. Polymorphisms in the cytochrome P450 enzyme (CYP2C9) that metabolizes warfarin are also known. In 2010, the U.S. Food and Drug Administration added a genotype-based dose table to the warfarin label (package insert). The influence of genetics on an individual’s response to drugs is known as pharmacogenetics.
D. Pathways
Three distinct pathways are involved in formation of the fibrin meshwork: the extrinsic pathway, the intrinsic pathway, and the common pathway. Production of FXa by the extrinsic and intrinsic pathways initiates the common pathway (see Fig. 35.2).