Problem A problem of bleeding cows and chickens rat poison and patients with thrombosis

During the 1920s a new disease of cattle involving fatal bleeding appeared over a wide area of prairie land in North America. It was eventually traced to feeding the animals on hay made from sweet clover (Melilotus alba and M. officinalis), which had 'mysteriously gone bad'. After eating the hay, the animals suffered a gradual decline in the clotting power of the blood (over about 15 days), followed by the development of internal haemorrhage which was generally fatal after 30—50 days. The disease was caused only by feeding the animals on spoilt sweet clover hay, and is commonly called 'sweet clover disease'. It could be treated by removing the spoilt hay from the animals' diet and transfusing them with blood freshly drawn from normal healthy animals.

What conclusions can you draw from these observations?

The addition of oxalate or citrate to blood will chelate calcium ions, and prevent clotting. Addition of a solution of calcium chloride to oxalated normal plasma results in rapid clotting; addition of calcium chloride to oxalated plasma from calves with sweet clover disease did not result in clotting.

What conclusions can you draw from this observation?

Partially purified prothrombin was prepared from blood of either healthy animals or those with sweet clover disease and tested on blood from an affected animal — the results are shown in Table 9.12.

What conclusions can you draw from these results?

The toxin present in spoiled sweet clover hay was identified as bis-hydroxycoumarin (dicoumarol; see Figure 11.10); it is formed by the oxidation of coumarin, which is naturally present in sweet clover. Once dicoumarol was available in adequate amounts, its mode of action could be investigated; early studies showed that there was a dose-dependent impairment of blood clotting, but with a latent period of 12—24 hours before any effect became apparent.

What conclusions can you draw from this observation?

Why do you think it was that dicoumarol was adopted for use in low doses to reduce blood clotting in patients at risk of thrombosis and as a rat poison in relatively high doses.

Further research at the University of Wisconsin resulted in the synthesis of a variety of compounds related to dicoumarol that were more potent as rat poisons and had fewer side-effects in human beings. The most successful of these compounds was called warfarin (from the initials of the Wisconsin Alumnus Research Fund, which supported the research — see Figure 11.10); it is still the most widely used anticoagulant in clinical use and the most commonly used rodenticide.

In 1929, Dam and co-workers in Copenhagen fed chickens on a fat-free diet and reported the development of subcutaneous and intramuscular haemorrhages, as well as impaired blood clotting. They went on to demonstrate a fat-soluble compound in vegetables, grains and animal liver that would normalize blood clotting when fed to deficient chickens. They proposed the name vitamin K (section 11.5) for this new nutrient, and defined a unit of biological activity as that amount required per kilogram body weight on three successive days to normalize blood clotting in a deficient animal.

Why do you think the response to vitamin K in deficient animals takes several days to develop?

In 1952, an army recruit attempted suicide by taking rat poison containing warfarin. On admission to hospital he had numerous subcutaneous haemorrhages and was suffering from nose-bleeds. His prothrombin time was 54 seconds, compared with a normal value of 14 seconds. (The prothrombin time is the time taken for the formation of a fibrin clot in citratedplasma after the addition of calcium ions and thromboplastin to activate

Table 9.12 Clotting time of blood from a calf affected by sweet clover disease when treated with the prothrombin fraction from a normal animal or an affected animal

Prothrombin fraction prepared from plasma of

Normal calf

Affected calf

Volume added (mL) Time to clot (min)

0.5 1.0 60 45

2.5 0.5 20 > 360

From data reported by Roderick LM (1931) American Journal of Physiology 96: 413—425.

From data reported by Roderick LM (1931) American Journal of Physiology 96: 413—425.

the extrinsic clotting system.) He was given 20 mg vitamin K intravenously daily for 10 days, when he recovered, with a normal prothrombin time.

What does this suggest about the way in which warfarin affects blood clotting?

In animals treated with warfarin it is possible to isolate a protein that reacts with antiserum to prothrombin, but has no biological activity. During warfarin treatment, the concentration of this abnormal, inactive, prothrombin in plasma increases, and that of active prothrombin decreases. The abnormal prothrombin is less negatively charged than normal prothrombin, and does not migrate so rapidly towards the anode on electrophoresis. The addition of calcium ions to a sample of normal prothrombin reduces its electrophoretic mobility but has no effect on the electrophoretic mobility of the abnormal prothrombin. Feeding high intakes of vitamin K to warfarin-treated animals normalizes their blood clotting and leads to disappearance of the abnormal prothrombin and reappearance of active prothrombin with normal calcium-binding capacity.

What conclusions can you draw from these results?

The results of studies of the synthesis of prothrombin in the post-mitochondrial supernatant fraction from liver of vitamin K-deficient rats incubated with vitamin K added in vitro are shown in Table 9.13.

What conclusions can you draw from these results?

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.

All of the codons for amino acids are known. How does y-carboxyglutamate become incorporated into prothrombin? What is the likely role of vitamin K in this process?

It has been known for some years that vitamin K must be reduced to its hydroquinone for activity in prothrombin synthesis, i.e. it is the hydroquinone that is the active metabolite of the vitamin. In rats treated with warfarin and given 14C-

Table 9.13 Prothrombin synthesis by post-mitochondrial supernatant fraction from liver of vitamin K-deficient rats

Relative prothrombin Incubation conditions activity (%)

No vitamin K 7 ± 1

  • 20 ^g/mL vitamin K 100
  • 20 ^.g/mL vitamin K+ cycloheximide 94 ± 1
  • 20 ^.g/mL vitamin K+ 2-chlorophytylmenaquinone 18 ± 1
  • 20 ^.g/mL vitamin K+ warfarin 87 ± 3
  • 20 ^.g/mL vitamin K, anaerobic 0

From data reported by Shah DV and Suttie JW (1974) Biochemical and Biophysical Research Communications 60: 1397-1402.

labelled vitamin K more than 50% of the radioactivity was found in a new compound, vitamin K epoxide, which has the biological activity of vitamin K in deficient animals. A very small amount is present in the liver of normal animals.

Can you propose the sequence of reactions which vitamin K undergoes in the synthesis of prothrombin?

Which step is likely to be inhibited by warfarin?

Can you explain why it is that high intakes of vitamin K will overcome the inhibition of prothrombin synthesis caused by warfarin?

The classical way of assessing vitamin K nutritional status was by determination of prothrombin time. Can you suggest a more sensitive way of detecting marginal vitamin K deficiency?

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Responses

  • daniela
    Are chickens fed rat poison?
    7 years ago
  • nadine
    Why is calf with sweet clover diseased did not result in clotting?
    1 year ago
  • ROGELIO MESSER
    Why is calf with sweet clover disease did not result in clotting?
    10 months ago

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