EXPERIMENTAL APPROACHES TO THE 
STUDY OF VITAMIN K 
Walter H. Seegers* 
This is a commentary on animal experiments related 
to vitamin K. The rich literature on this subject began 
to appear in 1929 with a description of dietary experi- 
ments in which chickens were used as test animals to 
see whether they could live without receiving choles- 
terol. They developed a bleeding tendency which was 
found to be due to a dietary deficiency of a vitamin not 
previously recognized. Without vitamin K, the plasma 
prothrombin concentration diminished and the blood 
coagulation time was prolonged. Even before vitamin K 
was discovered, cattle had been found to develop fatal 
hemorrhages when fed spoiled sweet clover hay. The 
toxic hay was later deliberately produced; the toxin was 
isolated (Dicumarol), synthesized, and marketed as an 
anticoagulant which functioned as an antagonist to vi- 
tamin K. Rabbits were used as test animals, and many 
were found to be resistant to the toxin. Dogs developed 
.vitamin K deficiency after surgical modifications which 
diverted bile via the urine, but the dog was mainly use- 
ful for studies on Dicumarol-vitamin K antagonism. 
With the successful isolation of plasma prothrombin, it 
was possible to build antibodies to the protein, and by 
using the fluorescent antibody technique demonstrate 
interesting aspects of prothrombin synthesis in the liver. 
With rats that were receiving Dicumarol, it was possible 
to make observations on stress. Various stressors, in- 
cluding some of a psychological nature, triggered inter- 
nal hemorrhages that were fatal. 
INTRODUCTION** 
The new and revised edition of Experiments 
on Animals by Paget appeared in 1903.^ Its 
mission was to point out the value of advances 
in biology made by experiments which required 
animals. I find it interesting that Lord Lister, 
who wrote the introduction, was the one who 
emphasized that surfaces are important for 
promoting the coagulation of blood. Today it is 
easy to recognize the importance of his observa- 
tions and additionally to outline some of the 
* Department of Physiology, Thrombosis Specialized Center of Re- 
search, Wayne State University School of Medicine, Detroit, Michi- 
Kan. 
** This work was supported by research grant HE-03424-1B from 
the Heart and Lung Institute, National Institutes of Health, U. S. 
Public Health Service. 
main enzyme reactions which bring about the 
sol-gel transformation. In order to appreciate 
v^ork on vitamin K, it is helpful to indicate 
briefly the nature of the chemistry of blood co- 
agulation which is understood to date (Fig- 
ure 1). 
Three basic reactions occur in the given 
order : 
(1) Formation of autoprothrombin C (Fac- 
tor Xa) 
(2) Formation of thrombin 
(3) Formation of fibrin 
Thrombin is required for the formation of 
fibrin and the limited proteolysis by this enzyme 
is quite specific, exact, unique and rather spe- 
cial. The formation of thrombin requires only 
autoprothrombin C, but the enzyme needs to 
have accessory substances to determine its spec- 
ificity. By itself, it degrades prothrombin ran- 
domly. However, with plasma Ac-globulin, the 
right kind of phospholipid, and calcium ions, 
thrombin is formed specifically. The formation 
of autoprothrombin C is a more general phe- 
nomenon. During clotting there is only frac- 
tional utilization, and much remains in serum. 
Several substances, and combinations of sub- 
stances, accelerate autoprothrombin C forma- 
tion. Among these substances are tissue ex- 
tracts, which have a high degree of species 
specificity. 
It is important for our discussion to appre- 
ciate that the two main enzjrme precursors, 
namely, autoprothrombin III (Factor X) and 
prothrombin, depend upon vitamin K for their 
synthesis in the liver. In the cir/2ulating blood, 
the two enzymes are bound together in the 
prothrombin complex. 
Avian blood became a favorite fluid for in- 
vestigation because it can be drawn into paraffin- 
lined glass vessels and, with good luck, will re- 
493 
