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Title: The effects of oral anticoagulants on the synthesis of glycoproteins
Author: Prowse, C. V.
ISNI:       0000 0001 3502 4721
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1976
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The coumarin related drugs have been used as oral anticoagulants for many years. These drugs help in the prevention of thrombotic episodes by reducing the activity of certain plasma proteins required for the clotting process. Administration of vitamin K reverses the effects of the coumarin drugs and deficiency of this vitamin results in similar symptoms as are induced by coumarin therapy. It has been proposed, therefore, that vitamin K and the coumarins act at a similar site and affect the plasma activity of the clotting proteins. Factors II, VII, IX and X. Study of the synthesis of these proteins has shown that the site of vitamin K (and coumarin) action is post-ribosomal and that the vitamin might be responsible for the introduction of a 'prosthetic' group into the proteins. This is supported by the finding that the plasma of coumarin treated patients, and cattle, contains a protein with the immuno logical characteristics of normal prothrombin but with negligible activity in the usual bioassay. In this study it has been shown that an abnormal prothrombin present in the plasma of patients undergoing anticoagulant therapy with Warfarin may be purified by absorption to barium citrate and subsequent chromatography on DEAE-Sephadex. This barium-absorbable coumarin prothrombin accounts for at least 70% of the immunological prothrombin in coumarin plasma, i.e. it is the major species of prothrombin during coumarin therapy. The abnormal protein is eluted from DEAE-Sephadex prior to normal prothrombin. Other workers in this field have concentrated on the barium non-absorbable prothrombin from cattle. This form of prothrombin has a lower biological activity than the barium-absorbable protein. Present theory explains this effect in terms of the inhibition, by the coumarin drugs, of the vitamin K-dependant carboxylation of ten glutamic acid residues in the N-terminal portion of the prothrombin with a resultant loss of barium and calcium binding ability. In order to investigate the reason for the low specific activity of the barium-absorbable prothrombin larger volumes of blood were required than it was feasible to obtain from outpatients. The corresponding protein was therefore isolated from the plasma of cows treated with 'therapeutic' levels of Warfarin. The bovine protein, like the human barium-absorbable prothrombin, has a reduced biological activity. Analysis of acid hydrolysates of this protein showed that it had an identical carbohydrate and amino acid content to normal prothrombin. Investigation of the activation of the various forms of prothrombin showed that abnormal prothrombin, from both cattle and human patients, gives rise to thrombin which has the same specific activity as normal thrombin and that the rate of activation of abnormal prothrombin is reduced. Thus abnormal prothrombin does not interact normally with its activating enzyme, Factor Xa, in the presence or absence of Factor V or phospholipid, which act as accelerators of normal activation. Following this it was shown that abnormal prothrombin had a reduced ability to bind calcium; such a defect accounts for the above activation differences. This defect was located in the N-terminal (Fragment 1) portion of the prothrombin molecule. Alkaline hydrolysis of the whole prothrombin molecule and of the isolated Fragment 1 showed that, in contrast to the normal protein, only about 6 of the possible ten glutamic acid residues were carboxylated in the barium-absorbable prothrombin. Detailed analysis of the N-terminal region of this protein was carried out by isolation and characterisation of peptides from this portion of the molecule and confirmed the view that this region is heterogenous in the abnormal protein. A further bovine plasma protein (Factor X3) which is affected by the administration of coumarin drugs and by vitamin K has also been partially characterised. This protein contains gamma-carboxy glutamic acid residues and is of similar size to Factors VII, IX and X, but has no clotting activity. Nor has any other activity been associated with Factor X3. Factor X is also affected by coumarin therapy. However in this case the protein exists as two formsr even in the vitamin K replete state. Despite this it was shown that these two forms, Factor X1 and Factor X2, differed in the extent of carboxylation. Factor X1 and X2 have different specific activities. As in the case of prothrombin this may be explicable in terms of a low gamma-carboxy glutamic acid content resulting in a reduced calcium binding ability. These results are used in proposing a model of vitamin K action which is discussed in terms of the possible effects of the coumarin drugs of proteins other than those involved in blood coagulation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available