Blood Coagulation Proteins

The principal physiological function that led to the discovery of vitamin K, and its confirmation as an essential vitamin for higher vertebrates, was its unique role in the blood clotting cascade. This cascade comprises a complex series of linked proenzyme-to-enzyme conversions, which leads eventually to a fibrin clot (Figure 2). Central to this process is the activation by calcium of gamma-carboxylated glutamyl (Gla) residues in some of the members of the cascade series: factors VII, IX, and X and factor II (prothrombin). In addition, there is an inhibitory level of control by proteins C, S, and possibly Z. All seven of these Gla proteins have Gla clusters that interact specifically with calcium so as to alter their polypeptide conformations and to permit their interaction with other members of the coagulation cascade (by exposing a phospholipid-binding domain) and hence leading either to activation or to inhibition of individual components. The Gla moieties of these and indeed all the vitamin K-dependent Gla proteins are formed by a post-translational carboxylation reaction catalyzed by the single enzyme, 'carboxylase,' at the

Figure 2 Vitamin K-dependent clotting factors. Factors II (prothrombin), VII, IX, and X and proteins C and S are all Gla proteins. The functions of proteins C and S, shown in bold, are inhibitory to the clotting cascade, whereas the other factors all form part of the cascade mechanism.

endoplasmic reticulum sites of Gla protein synthesis. In the case of the blood coagulation proteins, the sole site of synthesis is the liver. Each carboxylated protein has a C-terminal 'propeptide' sequence that binds the carboxylase enzyme, and directs a coordinated series of carboxylations of the recipient gluta-myl residues, before the propeptide is removed and the fully carboxylated protein is then secreted into the extracellular space for transport into the plasma.

Vitamin K acts as the essential recycling cofactor (or cosubstrate) for all protein carboxylation, Gla-forming reactions (Figure 3). In its dihydro or quinol form, the vitamin reacts with molecular oxygen, thereby creating a highly reactive, high-energy carba-nion at the Glu site for insertion of carbon dioxide, creating a new Gla residue. This vitamin K quinol oxidation step provides the essential energy for the endothermic carboxylation step. The other product of the reaction is the epoxide of vitamin K, comprising a three-membered carbon-oxygen ring. Since the oxidized vitamin needs to be recycled back to the quinol form before the next protein carboxylation cycle, a two-stage reduction process ensues, forming first vitamin K quinone and then the original quinol (Figure 3). Both of these reduction steps can be catalyzed by the enzyme vitamin K epoxide reductase, which is linked to a dithiol-disulfide reducing couple and which is highly sensitive to inhibition by the coumarin class of drugs, of which warfarin (Figure 1) is the best known and most commonly used member. The reduction of the intermediate vitamin K quinone to its quinol form can also be catalyzed by another, NAD(P)H-dependent, quinone reductase that is warfarin resistant, and for this reason the inhibition of carboxylation by warfarin can be reversed or antagonized by large doses of vitamin K provided exogen-ously in its normal quinone form. A severe deficiency of vitamin K, or treatment with coumarin drugs (for the control of excessive blood clotting tendency in humans), results in prolonged clotting times that can be detected by the standardized 'one stage pro-thrombin time' test, in which citrated or oxalated (i.e., calcium-complexed) blood is treated with tissue factor plus additional calcium so as to initiate the clotting process. However, a much more sensitive test for mild vitamin K deficiency is the PIVKA test (Proteins Induced by Vitamin K Absence or Antagonism), which is an immunological enzyme-linked immunosorbent assay (ELISA) test that specifically recognizes undercarboxylated blood clotting proteins and particularly des-gamma-carboxy prothrombin.

Proteins C and S, and possibly also Z, function differently from the other Gla-containing blood clotting factors that are an integral part of the fibrin-forming cascade. Protein C has a regulatory role, inactivating factors V and VIII, and in conjunction with protein S it also acts as a cofactor to enhance the rate of fibrinolysis of blood clots in locations where they are unwanted and potentially harmful. The exact function of protein Z remains unresolved, although interactions with thrombin and factor X have been reported. Clearly, there is a delicate balance of

Peptidyl glutamate -(Glu)-

O2, CO2, Carboxylase

Peptidyl 7-carboxyglutamate -(Gla)-

Figure 3 Vitamin K oxidation-reduction cycle during Gla formation. Oxidation of vitamin K hydroquinone (reduced vitamin) to vitamin K epoxide by molecular oxygen provides the energy needed to drive the carboxylation of peptidyl-Glu to peptidyl-Gla (i.e., gamma-carboxy glutamate). The vitamin K epoxide is then recycled by reduction with dithiols in two stages. The first stage requires a reductase enzyme that is coumarin drug (e.g., warfarin) inhibitable. The second stage can be catalyzed by either of two reductases, one of which is NAD(P)H dependent and is not warfarin inhibited.

Figure 3 Vitamin K oxidation-reduction cycle during Gla formation. Oxidation of vitamin K hydroquinone (reduced vitamin) to vitamin K epoxide by molecular oxygen provides the energy needed to drive the carboxylation of peptidyl-Glu to peptidyl-Gla (i.e., gamma-carboxy glutamate). The vitamin K epoxide is then recycled by reduction with dithiols in two stages. The first stage requires a reductase enzyme that is coumarin drug (e.g., warfarin) inhibitable. The second stage can be catalyzed by either of two reductases, one of which is NAD(P)H dependent and is not warfarin inhibited.

pro- and anti-clot formation and removal activities among the vitamin K-dependent Gla proteins of the cascade, although the net effect of a deficiency of the vitamin or of its antagonism by drugs appears to be a reduction of the clotting tendency.

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