Preprothrombin

Prothrombin and several other proteins of the blood clotting system (Factors VII, IX and X, and proteins C and S) each contain between four and six y-carboxygluta-mate residues which chelate calcium ions and so permit the binding of the blood clotting proteins to membranes. In vitamin K deficiency or in the presence of warfarin, an abnormal precursor of prothrombin (preprothrombin) containing little or no y-carboxyglutamate, and incapable of chelating calcium, is released into the circulation. 

Prothrombin normally contains 10 y-carboxyglutamate residues in the amino terminal region. In the presence of high concentrations of warfarin, a completely uncarboxylated precursor, preprothrombin, is released into the circulation. Before the nature of this precursor protein was known, it was called protein induced by vitamin K absence or antagonism (PIVKA), a term that is sometimes still used. 

At lower doses of anticoagulant, a variety of partially carboxylated preprothrombins are formed. Sequencing of these proteins shows that the... 

The response of deficient animals to repletion with vitamin K, or the administration of the vitamin after anticoagulants, is more rapid than might be expected. There is a considerable intracellular accumulation of preprothrombin, whichis immediately available for carboxylation when vitamin Kbecomes available. 

Preprothrombin can be determined immunologically, either using antiprothrombin antibodies, after adsorption of the y -carboxylated protein onto barium carbonate or using anti-preprothrombin antibodies that do not cross-react with prothrombin. Circulating concentrations of preprothrombin in vitamin K deficiency are of the order of 150 to 1,500 nmol per L. If elevated preprothrombin is because of vitamin K deficiency, then it will fall on administration of the vitamin, whereas if it is the result of liver disease, then vitamin K supplements will have no effect. 

In deficiency, there is also undercarboxylated osteocalcin in the circulation, and this provides a more sensitive index of marginal status it is detectable, and responds to supplements of vitamin K, in subjects with normal clotting time and no circulating preprothrombin (Sokoll and Sadowski, 1996 Binkley et al., 2000). 

The determination of vitamin K requirements is complicated by the intestinal bacterial synthesis of menaquinones and the extent to which these are absorbed and utilized (Section 5.1). Prolonged use of antibiotics leads to impaired blood clotting, but simple dietary restriction of vitamin K results in prolonged prothrombin time and increased circulating preprothrombin so it is apparent that bacterial synthesis is inadequate to meet requirements in the absence of a dietary intake of phylloquinone. Preprothrombin is elevated at intakes between 40 to 60 /xg per day, but not at intakes above 80 /rg per day (Suttie etal., 1988). 

The resultant modified E residues are gamma-carboxyglutamate (gla). This process is most clearly understood for factor II, also called preprothrombin. Prothrombin is modified pre-prothrombin. The gla residues are effective calcium ion chelators. Upon chelation of calcium, prothrombin interacts with phospholipids in membranes and is proteolysed to thrombin through the action of activated factor X (Xa). Dining the carboxylation reaction reduced hydroquinone form of vitamin K is converted to a 2,3-epoxide form. The regeneration of the hydroquinone form requires an uncharacterized reductase. This latter reaction is the site of action of the dicumarol-based anticoagulants such as warfarin. 

A novel amino acid is present in prothrombin that is not present in the abnormal prothrombin (now called preprothrombin) formed in vitamin K deficiency or on treatment with warfarin—y-carboxyglutamate (abbreviated to Gla see Figure 11.11). There are 10 Gla residues in the amino-terminal region of active prothrombin, and these form a binding site for four calcium ions. 

The main way of determining vitamin K status, and monitoring the efficacy of anticoagulant therapy, is by measuring the time required for the formation of a fibrin clot in citrated blood plasma after the addition of calcium ions and thromboplastin — the prothrombin time. A more sensitive index is provided by direct measurement of preprothrombin in plasma, most commonly by immunoassay using antisera against preprothrombin that do not react with prothrombin. 

Based on determination of clotting time, and direct measurement of prothrombin and preprothrombin, an intake of 1 Jg per kg body weight per day is considered adequate this forms the basis of reference intakes of between 65 and 80 Jg/day for adults. 

---